Transgenic plants with enhanced agronomic traits

ABSTRACT

This invention provides transgenic plant cells with recombinant DNA for expression of proteins that are useful for imparting enhanced agronomic trait(s) to transgenic crop plants. This invention also provides transgenic plants and progeny seed comprising the transgenic plant cells where the plants are selected for having an enhanced trait selected from the group of traits consisting of enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil. Also disclosed are methods for manufacturing transgenic seed and plants with enhanced traits.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of and claims the benefit of priority under 35 U.S.C. §120 to U.S. patent application Ser. No. 12/322,120, entitled “TRANSGENIC PLANTS WITH ENHANCED AGRONOMIC TRAITS,” filed on Jan. 29, 2009, which claims the benefit of priority under 35 USC §119(e) to U.S. provisional application Ser. Nos. 61/024,667, filed Jan. 30, 2008, 61/035,103, filed Mar. 10, 2008, 61/047,017, filed Apr. 22, 2008, 61/048,708, filed Apr. 29, 2008, and 61/078,027, filed Jul. 3, 2008, each of which is incorporated herein by reference in its entirety.

INCORPORATION OF SEQUENCE LISTING

Two copies of the sequence listing (Copy 1 and Copy 2) and a computer readable form (CRF) of the sequence listing, all non CD-Rs, each containing the text file named “55591_B_seq_listing.txt”, which is 74,407,936 bytes (measured in MS-WINDOWS), were created on Jan. 27, 2009, and filed Jan. 29, 2009, for the application accorded Ser. No. 12/322,120; and the sequence listing file “55591_B_seq_listing corrected.txt” which is 74,411,868 bytes and was created on Mar. 16, 2010 as well as the material on replacement CDs (Copy #1 Replacement and Copy #2 Replacement) named “Sequence,” file name “3126019US1.txt,” file size 74,411,868 bytes created on Aug. 9, 2010, and filed Aug. 9, 2010, for the application accorded Ser. No. 12/322,120, are incorporated herein by reference.

Incorporation of Large Table

Two copies of a large table (Copy 1 and Copy 2) containing a folder “pfamdir” on CD-Rs are incorporated herein by reference in their entirety. Folder “pfamdir” contains 103 Pfam Hidden Markov Models. The CD-Rs were created on Jan. 27, 2009, having a total size of 7,665,424 bytes (measured in MS-WINDOWS), and filed on Jan. 29, 2009, for the application accorded Ser. No. 12/322,120 and incorporated herein by reference.

FIELD OF THE INVENTION

Disclosed herein are recombinant DNA useful for providing enhanced traits to transgenic plants, seeds, pollen, plant cells and plant nuclei of such transgenic plants, methods of making and using such recombinant DNA, plants, seeds, pollen, plant cells and plant nuclei. Also disclosed are methods of producing hybrid corn seed comprising such recombinant DNA. All genetic resources disclosed herein were directly obtained from sources that are currently common to the United States; the ancestral sources of each specific genetic material is unknown.

SUMMARY OF THE INVENTION

An aspect of this invention provides recombinant DNA constructs comprising polynucleotides characterized by an encoded protein having amino acids representing a protein family domain module as described in Table 10. Another aspect of this invention provides recombinant DNA constructs comprising polynucleotides characterized by an encoded protein with an amino acid sequence that is at least 90% identical to a corresponding consensus sequence defined in table 8. Yet another aspect of this invention provides recombinant DNA constructs comprising polynucleotides characterized by reference to SEQ ID NO:1-121 and the cognate proteins with amino acid sequences having reference to SEQ ID NO:122-242. The recombinant DNA constructs are useful for providing enhanced traits when stably integrated into the chromosomes and expressed in the nuclei of transgenic plants cells. In some aspects of the invention the recombinant DNA constructs, when expressed in a plant cell, provide for expression of cognate proteins. In those aspects of the invention, the recombinant DNA constructs for expressing cognate proteins are characterized by cognate amino acid sequences having a sequence selected from SEQ ID NOs: 123-140, 145-151, 153-173, 175-187, 189, 191-202, 204-207, 209-238, and 242; having at least 95% identity over at least 95% of the length of a sequence selected from the group consisting of SEQ ID NOs: 123-140, 145-151, 153-173, 175-187, 189, 191-202, 204-207, 209-238, and 242 or that are homologous to a sequence selected from the group consisting of SEQ ID NOs: 123-140, 145-151, 153-173, 175-187, 189, 191-202, 204-207, 209-238, and 242.

In other aspects of the invention the recombinant DNA constructs provide for suppression of a native protein. In those other aspects of the invention the recombinant DNA constructs are characterized as being constructed with sense-oriented and anti-sense-oriented polynucleotides, e.g. polynucleotides derived from genes having SEQ ID NOs: 1, 20-23, 31, 53, 67, 69, 82, 87, and 118-120 or homologous genes. When the recombinant DNA construct is expressed in corn plants, the endogenous protein is a corn protein with an amino acid sequence of SEQ ID NO:141-144, 152, 174, 190, 203, 208, or 239-240 or the corn homolog of SEQ ID NOs:122, 188, or 241; when the recombinant DNA construct is expressed in soybean plants, the endogenous protein is a soybean protein with an amino acid sequence of SEQ ID NO: 122, 188, or 241 or is a soybean homolog of SEQ ID NOs: 141-144, 152, 174, 190, 203, 208, or 239-240; and when the recombinant DNA construct is expressed in a plant other than a corn or a soybean plant, the endogenous protein is the other plant's endogenous protein that has an amino acid sequence homologous to SEQ ID NO: 122, 141-144, 152, 174, 188, 190, 203, 208, or 239-241.

In practical aspects of this invention the recombinant DNA constructs of the invention are stably integrated into the chromosome of a plant cell nucleus.

This invention also provides transgenic plant cells comprising the stably integrated recombinant DNA constructs of the invention, transgenic plants and seeds comprising a plurality of such transgenic plant cells and transgenic pollen of such plants. Such transgenic plants are selected from a population of transgenic plants regenerated from plant cells transformed with recombinant DNA constructs by screening transgenic plants for an enhanced trait as compared to control plants. The enhanced trait is one or more of enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil.

In another aspect of the invention the plant cells, plants, seeds, and pollen further comprise DNA expressing a protein that provides tolerance from exposure to an herbicide applied at levels that are lethal to a wild type plant cell.

This invention also provides methods for manufacturing non-natural, transgenic seed that can be used to produce a crop of transgenic plants with an enhanced trait resulting from expression of a stably-integrated recombinant DNA construct. More specifically, the method comprises (a) screening a population of plants for an enhanced trait and a recombinant DNA construct, where individual plants in the population can exhibit the trait at a level less than, essentially the same as or greater than the level that the trait is exhibited in control plants, (b) selecting from the population one or more plants that exhibit the trait at a level greater than the level that said trait is exhibited in control plants, (c) collecting seed from a selected plant, (d) verifying that the recombinant DNA is stably integrated in said selected plants, (e) analyzing tissue of a selected plant to determine the production or suppression of a protein having the function of a protein encoded by nucleotides in a sequence of one of SEQ ID NOs:1-121. In one aspect of the invention, the plants in the population further comprise DNA expressing a protein that provides tolerance to exposure to a herbicide applied at levels that are lethal to wild type plant cells and the selecting is affected by treating the population with the herbicide, e.g. a glyphosate, dicamba, or glufosinate compound. In another aspect of the invention the plants are selected by identifying plants with the enhanced trait. The methods are especially useful for manufacturing corn, soybean, cotton, canola, alfalfa, wheat, rice, sugarcane or sugar beet seed.

Another aspect of the invention provides a method of producing hybrid corn seed comprising acquiring hybrid corn seed from a herbicide tolerant corn plant which also has stably-integrated, recombinant DNA construct comprising a promoter that is (a) functional in plant cells and (b) is operably linked to DNA that encodes or suppresses a protein having the function of a protein encoded by nucleotides in a sequence of one of SEQ ID NOs:1-121. The methods further comprise producing corn plants from said hybrid corn seed, wherein a fraction of the plants produced from said hybrid corn seed is homozygous for said recombinant DNA, a fraction of the plants produced from said hybrid corn seed is hemizygous for said recombinant DNA, and a fraction of the plants produced from said hybrid corn seed has none of said recombinant DNA; selecting corn plants which are homozygous and hemizygous for said recombinant DNA by treating with an herbicide; collecting seed from herbicide-treated-surviving corn plants and planting said seed to produce further progeny corn plants; repeating the selecting and collecting steps at least once to produce an inbred corn line; and crossing the inbred corn line with a second corn line to produce hybrid seed.

Another aspect of the invention provides a method of selecting a plant comprising plant cells of the invention by using an immunoreactive antibody to detect the presence or absence of protein expressed or suppressed by recombinant DNA in seed or plant tissue. Yet another aspect of the invention provides anti-counterfeit milled seed having, as an indication of origin, plant cells of this invention.

Still other aspects of this invention relate to transgenic plants with enhanced water use efficiency or enhanced nitrogen use efficiency. For instance, this invention provides methods of growing a corn, cotton, soybean, or canola crop without irrigation water comprising planting seed having plant cells of the invention which are selected for enhanced water use efficiency. Alternatively methods comprise applying reduced irrigation water, e.g. providing up to 300 millimeters of ground water during the production of a corn crop. This invention also provides methods of growing a corn, cotton, soybean or canola crop without added nitrogen fertilizer comprising planting seed having plant cells of the invention which are selected for enhanced nitrogen use efficiency.

Another aspect of the invention provides a mixture comprising plants cells and an antibody to a protein produced in the cells where the protein has an amino acid sequence that has at least 95% identity over at least 95% of the length of a reference sequence selected from the group consisting of SEQ ID NO: 122-242 when the sequence is aligned to the reference sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-4 are plasmid maps.

DETAILED DESCRIPTION OF THE INVENTION

In the attached sequence listing:

SEQ ID NO:1-121 are nucleotide sequences of the coding strand of DNA for “genes” used in the recombinant DNA imparting an enhanced trait in plant cells, i.e. each represents a coding sequence for a protein;

SEQ ID NO: 122-242 are amino acid sequences of the cognate protein of the “genes” with nucleotide coding sequences 1-121;

SEQ ID NO: 243-17376 are amino acid sequences of homologous proteins;

SEQ ID NO: 17377 is a nucleotide sequence of a base plasmid vector useful for corn transformation;

SEQ ID NO: 17378 is a nucleotide sequence of a base plasmid vector useful for soybean and canola transformation;

SEQ ID NO: 17379 is a nucleotide sequence of a base plasmid vector useful for cotton transformation;

SEQ ID NO: 17380 is a nucleotide sequence of a base plasmid vector useful for co-transformation to produce gene stacks in corn;

SEQ ID NO: 17381-17402 are consensus sequences.

Table 8 lists the protein SEQ ID NOs and their corresponding consensus SEQ ID NOs.

As used herein a “plant cell” means a plant cell that is transformed with stably-integrated, non-natural, recombinant DNA, e.g. by Agrobacterium-mediated transformation or by bombardment using microparticles coated with recombinant DNA or other means. A plant cell of this invention can be an originally-transformed plant cell that exists as a microorganism or as a progeny plant cell that is regenerated into differentiated tissue, e.g. into a transgenic plant with stably-integrated, non-natural recombinant DNA, or seed or pollen derived from a progeny transgenic plant.

As used herein a “transgenic plant” means a plant whose genome has been altered by the stable integration of recombinant DNA. A transgenic plant includes a plant regenerated from an originally-transformed plant cell and progeny transgenic plants from later generations or crosses of a transformed plant.

As used herein “recombinant DNA” means DNA which has been a genetically engineered and constructed outside of a cell including DNA containing naturally occurring DNA or cDNA or synthetic DNA.

As used herein “consensus sequence” means an artificial sequence of amino acids in a conserved region of an alignment of amino acid sequences of homologous proteins, e.g. as determined by a CLUSTALW alignment of amino acid sequence of homolog proteins.

As used herein a “homolog” means a protein in a group of proteins that perform the same biological function, e.g. proteins that belong to the same Pfam protein family and that provide a common enhanced trait in transgenic plants of this invention. Homologs are expressed by homologous genes. With reference to homologous genes, homologs include orthologs, i.e. genes expressed in different species that evolved from a common ancestral genes by speciation and encode proteins retain the same function, but do not include paralogs, i.e. genes that are related by duplication but have evolved to encode proteins with different functions. Homologous genes include naturally occurring alleles and artificially-created variants. Degeneracy of the genetic code provides the possibility to substitute at least one base of the protein encoding sequence of a gene with a different base without causing the amino acid sequence of the polypeptide produced from the gene to be changed. When optimally aligned, homolog proteins have at least 60% identity, more preferably about 65% or higher, more preferably about 70% or higher, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90% identity, more preferably at least 95, 96, 97, 98, or 99% identity over the full length of a protein identified as being associated with imparting an enhanced trait when expressed in plant cells. In one aspect of the invention homolog proteins have an amino acid sequence that has at least 90% identity to a consensus amino acid sequence of proteins and homologs disclosed herein.

Homologs are identified by comparison of amino acid sequence, e.g. manually or by use of a computer-based tool using known homology-based search algorithms such as the suite of BLAST programs available from NCBI. A local sequence alignment program, e.g. BLAST, can be used to search a database of sequences to find similar sequences, and the summary Expectation value (E-value) used to measure the sequence base similarity. Because a protein hit with the best E-value for a particular organism may not necessarily be an ortholog, i.e. have the same function, or be the only ortholog, a reciprocal query is used to filter hit sequences with significant E-values for ortholog identification. The reciprocal query entails search of the significant hits against a database of amino acid sequences from the base organism that are similar to the sequence of the query protein. A hit can be identified as an ortholog, when the reciprocal query's best hit is the query protein itself or a protein encoded by a duplicated gene after speciation. A further aspect of the homologs encoded by DNA useful in the transgenic plants of the invention are those proteins that differ from a disclosed protein as the result of deletion or insertion of one or more amino acids in a native sequence.

Percent identity describes the extent to which the sequences of DNA or protein segments are invariant in an alignment of sequences, for example nucleotide sequences or amino acid sequences. An alignment of sequences is created by manually aligning two sequences, e.g. a stated sequence, as provided herein, as a reference, and another sequence, to produce the highest number of matching elements, e.g. individual nucleotides or amino acids, while allowing for the introduction of gaps into either sequence. An “identity fraction” for a sequence aligned with a reference sequence is the number of matching elements, divided by the full length of the reference sequence, not including gaps introduced by the alignment process into the reference sequence. “Percent identity” (“% identity”) as used herein is the identity fraction times 100.

“Pfam” is a large collection of multiple sequence alignments and hidden Markov models covering many common protein families, e.g. Pfam version 19.0 (December 2005) contains alignments and models for 8183 protein families and is based on the Swissprot 47.0 and SP-TrEMBL 30.0 protein sequence databases. See S. R. Eddy, “Profile Hidden Markov Models”, Bioinformatics 14:755-763, 1998. The Pfam database is currently maintained and updated by the Pfam Consortium. The alignments represent some evolutionary conserved structure that has implications for the protein's function. Profile hidden Markov models (profile HMMs) built from the protein family alignments are useful for automatically recognizing that a new protein belongs to an existing protein family even if the homology by alignment appears to be low.

Protein domains are identified by querying the amino acid sequence of a protein against Hidden Markov Models which characterize protein family domains (“Pfam domains”) using HMMER software, which is available from the Pfam Consortium. The HMMER software is also disclosed in patent application publication US 2008/0148432 A1 incorporated herein by reference. A protein domain meeting the gathering cutoff for the alignment of a particular Pfam domain is considered to contain the Pfam domain.

A “Pfam domain module” is a representation of Pfam domains in a protein, in order from N terminus to C terminus. In a Pfam domain module individual Pfam domains are separated by double colons “::”. The order and copy number of the Pfam domains from N to C terminus are attributes of a Pfam domain module. Although the copy number of repetitive domains is important, varying copy number often enables a similar function. Thus, a Pfam domain module with multiple copies of a domain should define an equivalent Pfam domain module with variance in the number of multiple copies. A Pfam domain module is not specific for distance between adjacent domains, but contemplates natural distances and variations in distance that provide equivalent function. The Pfam database contains both narrowly- and broadly-defined domains, leading to identification of overlapping domains on some proteins. A Pfam domain module is characterized by non-overlapping domains. Where there is overlap, the domain having a function that is more closely associated with the function of the protein (based on the E value of the Pfam match) is selected.

Once one DNA is identified as encoding a protein which imparts an enhanced trait when expressed in transgenic plants, other DNA encoding proteins with the same Pfam domain module are identified by querying the amino acid sequence of protein encoded by candidate DNA against the Hidden Markov Models which characterizes the Pfam domains using HMMER software. Candidate proteins meeting the same Pfam domain module are in the protein family and have cognate DNA that is useful in constructing recombinant DNA for the use in the plant cells of this invention. Hidden Markov Model databases for use with HMMER software in identifying DNA expressing protein with a common Pfam domain module for recombinant DNA in the plant cells of this invention are included in the large table incorporated into this application.

The HMMER software and Pfam databases (version 19.0) were used to identify known domains in the proteins corresponding to amino acid sequence of SEQ ID NOs: 123-132, 134-135, 137, 139-140, 142, 145-152, 154, 156-172, 175-181, 183-196, 198-202, 204-205, 207, 209-211, 213, 216, 218-228, 230-233, 235, 238-239, 241-242. All DNA encoding proteins that have scores higher than the gathering cutoff disclosed in Table 11 by Pfam analysis disclosed herein can be used in recombinant DNA of the plant cells of this invention, e.g. for selecting transgenic plants having enhanced agronomic traits. The relevant Pfarns modules for use in this invention, as more specifically disclosed below, are 14-3-3, 20G-FeII_Oxy, AhpC-TSA::1cysPrx_C, AP2, B3::Auxin_resp::AUX_IAA, Brix, CBFB_NFYA, CBFD_NFYB_HMF, Cellulose_synt, Copine, DnaJ, DUF231, DUF260, DUF296, DUF761, DUF778, E2F_TDP, efhand, FAR1::FAR1, F-box, Fer2::Fer2_(—)2::FAD_binding_(—)5::CO_deh_flav_C::Ald_Xan_dh_C::Ald_Xan_dh_C2, GATase_(—)2::Glu_syn_centrak:Glu_synthase::GXGXG, Gln-synt_N::Gln-synt_C, Globin, Glu_synthase, Glyco_hydro_(—)18, Glyco_transf 20::Trehalose_PPase, Glycos_transf_(—)1, GMC_oxred_N, GSHPx, HEAT::HEAT::FAT::Rapamycin_bind::PI3_PI4_kinase::FATC, HLH, HSP20, HSP70, LRRNT_(—)2::LRR_(—)1::LRR_(—)1::LRR_(—)1::Pkinase_Tyr, Mannitol_dh::Mannitol_dh_C, Na_H_Exchanger, NAPRTase, Nucleoside_tran, PALP, PAS_(—)2::GAF::Phytochrome::PAS, PAS_(—)2::GAF::Phytochrome::PAS::PAS::H isKA::HATPase_c, PAS_(—)3::PAS::Pkinase, PDZ::Peptidase_S41, Peptidase_S10, Peptidase_S51, PPR::PPR::PPR::PPR::PPR::PPR::PPR::PPR::PPR::PPR::PPR, PTR2, RAMP4, Response_reg, RimK::Mur_ligase_M::Mur_ligase_C, RNase_PH_C, SAM_(—)1::DRMBL, SAP::PHD::zf-MIZ, SAP::zf-MIZ, SNF2—N::Helicase_C, Spermine_synth, SRF-TF, SSF, Synaptobrevin, TP6A_N, TrkH, UDPGP, XRN_N::zf-CCHC, zf-A20::zf-AN1, and zf-B_box, for which databases are included in the appended computer listing.

As used herein “promoter” means regulatory DNA for initializing transcription. A “plant promoter” is a promoter capable of initiating transcription in plant cells whether or not its origin is a plant cell, e.g. is it well known that Agrobacterium promoters are functional in plant cells. Thus, plant promoters include promoter DNA obtained from plants, plant viruses and bacteria such as Agrobacterium and Bradyrhizobium bacteria. Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, or seeds. Such promoters are referred to as “tissue preferred”. Promoters that initiate transcription only in certain tissues are referred to as “tissue specific”. A “cell type” specific promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves. An “inducible” or “repressible” promoter is a promoter which is under environmental control. Examples of environmental conditions that may effect transcription by inducible promoters include anaerobic conditions, or certain chemicals, or the presence of light. Tissue specific, tissue preferred, cell type specific, and inducible promoters constitute the class of “non-constitutive” promoters. A “constitutive” promoter is a promoter which is active under most conditions.

As used herein “operably linked” means the association of two or more DNA fragments in a recombinant DNA construct so that the function of one, e.g. protein-encoding DNA, is controlled by the other, e.g. a promoter.

As used herein “expressed” means produced, e.g. a protein is expressed in a plant cell when its cognate DNA is transcribed to mRNA that is translated to the protein.

As used herein “suppressed” means decreased, e.g. a protein is suppressed in a plant cell when there is a decrease in the amount and/or activity of the protein in the plant cell. The presence or activity of the protein can be decreased by any amount up to and including a total loss of protein expression and/or activity.

As used herein a “control plant” means a plant that does not contain the recombinant DNA that imparts an enhanced trait. A control plant is used to identify and select a transgenic plant that has an enhanced trait. A suitable control plant can be a non-transgenic plant of the parental line used to generate a transgenic plant, i.e. devoid of recombinant DNA. A suitable control plant may in some cases be a progeny of a hemizygous transgenic plant line that does not contain the recombinant DNA, known as a negative segregant.

As used herein an “enhanced trait” means a characteristic of a transgenic plant that includes, but is not limited to, an enhance agronomic trait characterized by enhanced plant morphology, physiology, growth and development, yield, nutritional enhancement, disease or pest resistance, or environmental or chemical tolerance. In more specific aspects of this invention enhanced trait is selected from group of enhanced traits consisting of enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil. In an important aspect of the invention the enhanced trait is enhanced yield including increased yield under non-stress conditions and increased yield under environmental stress conditions. Stress conditions may include, for example, drought, shade, fungal disease, viral disease, bacterial disease, insect infestation, nematode infestation, cold temperature exposure, heat exposure, osmotic stress, reduced nitrogen nutrient availability, reduced phosphorus nutrient availability and high plant density. “Yield” can be affected by many properties including without limitation, plant height, pod number, pod position on the plant, number of internodes, incidence of pod shatter, grain size, efficiency of nodulation and nitrogen fixation, efficiency of nutrient assimilation, resistance to biotic and abiotic stress, carbon assimilation, plant architecture, resistance to lodging, percent seed germination, seedling vigor, and juvenile traits. Yield can also be affected by efficiency of germination (including germination in stressed conditions), growth rate (including growth rate in stressed conditions), ear number, seed number per ear, seed size, composition of seed (starch, oil, protein) and characteristics of seed fill.

Increased yield of a transgenic plant of the present invention can be measured in a number of ways, including test weight, seed number per plant, seed weight, seed number per unit area (i.e. seeds, or weight of seeds, per acre), bushels per acre, tons per acre, or kilo per hectare. For example, corn yield may be measured as production of shelled corn kernels per unit of production area, for example in bushels per acre or metric tons per hectare, often reported on a moisture adjusted basis, for example at 15.5 percent moisture. Increased yield may result from improved utilization of key biochemical compounds, such as nitrogen, phosphorous and carbohydrate, or from improved responses to environmental stresses, such as cold, heat, drought, salt, and attack by pests or pathogens. Recombinant DNA used in this invention can also be used to provide plants having improved growth and development, and ultimately increased yield, as the result of modified expression of plant growth regulators or modification of cell cycle or photosynthesis pathways. Also of interest is the generation of transgenic plants that demonstrate enhanced yield with respect to a seed component that may or may not correspond to an increase in overall plant yield. Such properties include enhancements in seed oil, seed molecules such as protein and starch, oil components as may be manifest by an alterations in the ratios of seed components.

Recombinant DNA constructs are assembled using methods well known to persons of ordinary skill in the art and typically comprise a promoter operably linked to DNA, the expression of which provides the enhanced agronomic trait. Other construct components may include additional regulatory elements, such as 5′ leaders and introns for enhancing transcription, 3′ untranslated regions (such as polyadenylation signals and sites), DNA for transit or signal peptides.

Numerous promoters that are active in plant cells have been described in the literature. These include promoters present in plant genomes as well as promoters from other sources, including nopaline synthase (NOS) promoter and octopine synthase (OCS) promoters carried on tumor-inducing plasmids of Agrobacterium tumefaciens and the CaMV35S promoters from the cauliflower mosaic virus as disclosed in U.S. Pat. Nos. 5,164,316 and 5,322,938. Useful promoters derived from plant genes are found in U.S. Pat. No. 5,641,876 which discloses a rice actin promoter, U.S. Pat. No. 7,151,204 which discloses a maize chloroplast aldolase promoter and a maize aldolase (FDA) promoter, and US Patent Application Publication 2003/0131377 A1 which discloses a maize nicotianamine synthase promoter. These and numerous other promoters that function in plant cells are known to those skilled in the art and available for use in recombinant polynucleotides of the present invention to provide for expression of desired genes in transgenic plant cells.

Furthermore, the promoters may be altered to contain multiple “enhancer sequences” to assist in elevating gene expression. Such enhancers are known in the art. By including an enhancer sequence with such constructs, the expression of the selected protein may be enhanced. These enhancers often are found 5′ to the start of transcription in a promoter that functions in eukaryotic cells, but can often be inserted upstream (5′) or downstream (3′) to the coding sequence. In some instances, these 5′ enhancing elements are introns. Particularly useful as enhancers are the 5′ introns of the rice actin 1 (see U.S. Pat. No. 5,641,876) and rice actin 2 genes, the maize alcohol dehydrogenase gene intron, the maize heat shock protein 70 gene intron (U.S. Pat. No. 5,593,874) and the maize shrunken 1 gene. See also US Patent Application Publication 2002/0192813A1 which discloses 5′, 3′ and intron elements useful in the design of effective plant expression vectors.

In other aspects of the invention, sufficient expression in plant seed tissues is desired to affect improvements in seed composition. Exemplary promoters for use for seed composition modification include promoters from seed genes such as napin as disclosed in U.S. Pat. No. 5,420,034, maize L3 oleosin as disclosed in U.S. Pat. No. 6,433,252), zein Z27 as disclosed by Russell et al. (1997) Transgenic Res. 6(2):157-166), globulin 1 as disclosed by Belanger et al (1991) Genetics 129:863-872), glutelin 1 as disclosed by Russell (1997) supra), and peroxiredoxin antioxidant (Per1) as disclosed by Stacy et al. (1996) Plant Mol Biol. 31(6):1205-1216.

Recombinant DNA constructs useful in this invention will also generally include a 3′ element that typically contains a polyadenylation signal and site. Well-known 3′ elements include those from Agrobacterium tumefaciens genes such as nos 3′, tml 3′, tmr 3′, tms 3′, ocs 3′, tr7 3′, for example disclosed in U.S. Pat. No. 6,090,627; 3′ elements from plant genes such as wheat (Triticum aesevitum) heat shock protein 17 (Hsp17 3′), a wheat ubiquitin gene, a wheat fructose-1,6-biphosphatase gene, a rice glutelin gene, a rice lactate dehydrogenase gene and a rice beta-tubulin gene, all of which are disclosed in US Patent Application Publication 2002/0192813 A1; and the pea (Pisum sativum) ribulose biphosphate carboxylase gene (rbs 3′), and 3′ elements from the genes within the host plant.

Constructs and vectors may also include a transit peptide for targeting of a gene to a plant organelle, particularly to a chloroplast, leucoplast or other plastid organelle. For descriptions of the use of chloroplast transit peptides see U.S. Pat. No. 5,188,642 and U.S. Pat. No. 5,728,925. For description of the transit peptide region of an Arabidopsis EPSPS gene useful in the present invention, see Klee, H. J. et al (MGG (1987) 210:437-442).

Recombinant DNA constructs for gene suppression can be designed for any of a number the well-known methods for suppressing transcription of a gene, the accumulation of the mRNA corresponding to that gene or preventing translation of the transcript into protein. Posttranscriptional gene suppression can be practically effected by transcription of RNA that forms double-stranded RNA (dsRNA) having homology to mRNA produced from a gene targeted for suppression.

Gene suppression can also be achieved by insertion mutations created by transposable elements may also prevent gene function. For example, in many dicot plants, transformation with the T-DNA of Agrobacterium may be readily achieved and large numbers of transformants can be rapidly obtained. Also, some species have lines with active transposable elements that can efficiently be used for the generation of large numbers of insertion mutations, while some other species lack such options. Mutant plants produced by Agrobacterium or transposon mutagenesis and having altered expression of a polypeptide of interest can be identified using the polynucleotides of the present invention. For example, a large population of mutated plants may be screened with polynucleotides encoding the polypeptide of interest to detect mutated plants having an insertion in the gene encoding the polypeptide of interest.

Transgenic plants may comprise a stack of one or more polynucleotides disclosed herein resulting in the production or suppression of multiple polypeptide sequences, Transgenic plants comprising stacks of polynucleotide sequences can be obtained by either or both of traditional breeding methods or through genetic engineering methods. These methods include, but are not limited to, breeding individual lines each comprising a polynucleotide of interest, transforming a transgenic plant comprising a gene disclosed herein with a subsequent gene, and co-transformation of genes into a single plant cell. Co-transformation of genes can be carried out using single transformation vectors comprising multiple genes or genes carried separately on multiple vectors.

Transgenic plants comprising or derived from plant cells of this invention transformed with recombinant DNA can be further enhanced with stacked traits, e.g. a crop plant having an enhanced trait resulting from expression of DNA disclosed herein in combination with herbicide and/or pest resistance traits. For example, genes of the current invention can be stacked with other traits of agronomic interest, such as a trait providing herbicide resistance, or insect resistance, such as using a gene from Bacillus thuringensis to provide resistance against lepidopteran, coliopteran, homopteran, hemiopteran, and other insects. Herbicides for which transgenic plant tolerance has been demonstrated and the method of the present invention can be applied include, but are not limited to, glyphosate, dicamba, glufosinate, sulfonylurea, bromoxynil and norflurazon herbicides. Polynucleotide molecules encoding proteins involved in herbicide tolerance are well-known in the art and include, but are not limited to, a polynucleotide molecule encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) disclosed in U.S. Pat. Nos. 5,094,945; 5,627,061; 5,633,435 and 6,040,497 for imparting glyphosate tolerance; polynucleotide molecules encoding a glyphosate oxidoreductase (GOX) disclosed in U.S. Pat. No. 5,463,175 and a glyphosate-N-acetyl transferase (GAT) disclosed in US Patent Application Publication 2003/0083480 A1 also for imparting glyphosate tolerance; dicamba monooxygenase disclosed in US Patent Application Publication 2003/0135879 A1 for imparting dicamba tolerance; a polynucleotide molecule encoding bromoxynil nitrilase (Bxn) disclosed in U.S. Pat. No. 4,810,648 for imparting bromoxynil tolerance; a polynucleotide molecule encoding phytoene desaturase (crtI) described in Misawa et al, (1993)Plant J. 4:833-840 and in Misawa et al, (1994) Plant J. 6; 481-489 for norflurazon tolerance; a polynucleotide molecule encoding acetohydroxyacid synthase (AHAS, aka ALS) described in Sathasiivan et al. (1990) Nucl. Acids Res. 18:2188-2193 for imparting tolerance to sulfonylurea herbicides; polynucleotide molecules known as bar genes disclosed in DeBlock, et al. (1987) EMBO J. 6:2513-2519 for imparting glufosinate and bialaphos tolerance; polynucleotide molecules disclosed in US Patent Application Publication 2003/010609 A1 for imparting N-amino methyl phosphonic acid tolerance; polynucleotide molecules disclosed in U.S. Pat. No. 6,107,549 for impartinig pyridine herbicide resistance; molecules and methods for imparting tolerance to multiple herbicides such as glyphosate, atrazine, ALS inhibitors, isoxoflutole and glufosinate herbicides are disclosed in U.S. Pat. No. 6,376,754 and US Patent Application Publication 2002/0112260. Molecules and methods for imparting insect/nematode/virus resistance are disclosed in U.S. Pat. Nos. 5,250,515; 5,880,275; 6,506,599; 5,986,175 and US Patent Application Publication 2003/0150017 A1.

Plant Cell Transformation Methods

Numerous methods for transforming chromosomes in a plant cell nucleus with recombinant DNA are known in the art and are used in methods of preparing a transgenic plant cell nucleus cell, and plant. Two effective methods for such transformation are Agrobacterium-mediated transformation and microprojectile bombardment. Microprojectile bombardment methods are illustrated in U.S. Pat. Nos. 5,015,580 (soybean); 5,550,318 (corn); 5,538,880 (corn); 5,914,451 (soybean); 6,160,208 (corn); 6,399,861 (corn); 6,153,812 (wheat) and 6,365,807 (rice) and Agrobacterium-mediated transformation is described in U.S. Pat. Nos. 5,159,135 (cotton); 5,824,877 (soybean); 5,463,174 (canola); 5,591,616 (corn); 5,846,797 (cotton); 6,384,301 (soybean), 7,026,528 (wheat) and 6,329,571 (rice), US Patent Application Publication 2004/0087030 A1 (cotton), and US Patent Application Publication 2001/0042257 A1 (sugar beet), all of which are incorporated herein by reference for enabling the production of transgenic plants. Transformation of plant material is practiced in tissue culture on a nutrient media, i.e. a mixture of nutrients that will allow cells to grow in vitro. Recipient cell targets include, but are not limited to, meristem cells, hypocotyls, calli, immature embryos and gametic cells such as microspores, pollen, sperm and egg cells. Callus may be initiated from tissue sources including, but not limited to, immature embryos, hypocotyls, seedling apical meristems, microspores and the like. Cells containing a transgenic nucleus are grown into transgenic plants.

In addition to direct transformation of a plant material with a recombinant DNA, a transgenic plant cell nucleus can be prepared by crossing a first plant having cells with a transgenic nucleus with recombinant DNA with a second plant lacking the transgenic nucleus. For example, recombinant DNA can be introduced into a nucleus from a first plant line that is amenable to transformation to transgenic nucleus in cells that are grown into a transgenic plant which can be crossed with a second plant line to introgress the recombinant DNA into the second plant line. A transgenic plant with recombinant DNA providing an enhanced trait, e.g. enhanced yield, can be crossed with transgenic plant line having other recombinant DNA that confers another trait, for example herbicide resistance or pest resistance, to produce progeny plants having recombinant DNA that confers both traits. Typically, in such breeding for combining traits the transgenic plant donating the additional trait is a male line and the transgenic plant carrying the base traits is the female line. The progeny of this cross will segregate such that some of the plants will carry the DNA for both parental traits and some will carry DNA for one parental trait; such plants can be identified by markers associated with parental recombinant DNA, e.g. marker identification by analysis for recombinant DNA or, in the case where a selectable marker is linked to the recombinant, by application of the selecting agent such as a herbicide for use with a herbicide tolerance marker, or by selection for the enhanced trait. Progeny plants carrying DNA for both parental traits can be crossed back into the female parent line multiple times, for example usually 6 to 8 generations, to produce a progeny plant with substantially the same genotype as one original transgenic parental line but for the recombinant DNA of the other transgenic parental line

In the practice of transformation DNA is typically introduced into only a small percentage of target plant cells in any one transformation experiment. Marker genes are used to provide an efficient system for identification of those cells that are stably transformed by receiving and integrating a recombinant DNA molecule into their genomes. Preferred marker genes provide selective markers which confer resistance to a selective agent, such as an antibiotic or a herbicide. Any of the herbicides to which plants of this invention may be resistant are useful agents for selective markers. Potentially transformed cells are exposed to the selective agent. In the population of surviving cells will be those cells where, generally, the resistance-conferring gene is integrated and expressed at sufficient levels to permit cell survival. Cells may be tested further to confirm stable integration of the exogenous DNA. Commonly used selective marker genes include those conferring resistance to antibiotics such as kanamycin and paromomycin (nptII), hygromycin B (aph IV), spectinomycin (aadA) and gentamycin (aac3 and aacC4) or resistance to herbicides such as glufosinate (bar or pat), dicamba (DMO) and glyphosate (aroA or EPSPS). Examples of such selectable markers are illustrated in U.S. Pat. Nos. 5,550,318; 5,633,435; 5,780,708 and 6,118,047. Markers which provide an ability to visually screen transformants can also be employed, for example, a gene expressing a colored or fluorescent protein such as a luciferase or green fluorescent protein (GFP) or a gene expressing a beta-glucuronidase or uidA gene (GUS) for which various chromogenic substrates are known.

Plant cells that survive exposure to the selective agent, or plant cells that have been scored positive in a screening assay, may be cultured in regeneration media and allowed to mature into plants. Developing plantlets regenerated from transformed plant cells can be transferred to plant growth mix, and hardened off, for example, in an environmentally controlled chamber at about 85% relative humidity, 600 ppm CO₂, and 25-250 microeinsteins m⁻² s⁻¹ of light, prior to transfer to a greenhouse or growth chamber for maturation. Plants are regenerated from about 6 weeks to 10 months after a transformant is identified, depending on the initial tissue, and plant species. Plants may be pollinated using conventional plant breeding methods known to those of skill in the art and seed produced, for example self-pollination is commonly used with transgenic corn. The regenerated transformed plant or its progeny seed or plants can be tested for expression of the recombinant DNA and selected for the presence of enhanced agronomic trait.

Transgenic Plants and Seeds

Transgenic plants derived from transgenic plant cells having a transgenic nucleus of this invention are grown to generate transgenic plants having an enhanced trait as compared to a control plant and produce transgenic seed and haploid pollen of this invention. Such plants with enhanced traits are identified by selection of transformed plants or progeny seed for the enhanced trait. For efficiency a selection method is designed to evaluate multiple transgenic plants (events) comprising the recombinant DNA, for example multiple plants from 2 to 20 or more transgenic events. Transgenic plants grown from transgenic seed provided herein demonstrate improved agronomic traits that contribute to increased yield or other trait that provides increased plant value, including, for example, improved seed quality. Of particular interest are plants having enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil. Table 1 provides a list of protein encoding DNA (“genes”) that are useful as recombinant DNA for production of transgenic plants with enhanced agronomic trait, the elements of Table 1 are described by reference to:

“PEP SEQ ID NO” identifies an amino acid sequence from SEQ ID NO: 122 to 242. “NUC SEQ ID NO” identifies a DNA sequence from SEQ ID NO:1 to 121. “Gene ID” refers to an arbitrary identifier. “Gene Name” denotes a common name for the protein encoded by the recombinant DNA preceded by the abbreviated genus and species as fully defined in the sequence listing. The + or − preceding the gene name indicates whether the protein is expressed (+) or suppressed (−) in plants to provide an enhanced trait. “Annotation” refers to a description of the top hit protein obtained from an amino acid sequence query of each PEP SEQ ID NO to GENBANK database of the National Center for Biotechnology Information (ncbi).

TABLE 1 NUC PEP SEQ ID SEQ ID NO NO Gene ID Gene Name Annotation 1 122 Mnom000132-Mnom000134 −Gm.GW2 emb|CAN66658.1|hypothetical protein [Vitis vinifera] 2 123 Mnom000202 +At.CGPG5239 gb|ABM06011.1|At1g67800 [Arabidopsis thaliana] 3 124 Mnom000215-Mnom000216 +Ta.NF-YA1 ref|NP_001048285.1|Os02g0776400 [Oryza sativa (japonica cultivar-group)] 4 125 Mnom000219-Mnom000220 +Zm.NF- ref|NP_001060230.1|Os07g0606600 [Oryza YB3/7/8 sativa (japonica cultivar-group)] 5 126 Mnom000256-Mnom000257 +Bs.GOGATII ref|NP_388541.1|hypothetical protein BSU06590 [Bacillus subtilis subsp. subtilis str. 168] 6 127 Mnom000260-Mnom000261 +Zm.TOR gb|AAW78347.1|target of rapamycin [Zea mays] 7 128 Mnom000262 +Zm.APO1 dbj|BAD45387.1|putative stamina pistilloidia [Oryza sativa (japonica cultivar-group)] 8 129 Mnom000324 +Gm.PTI4-3 AF245119_1 AP2-related transcription factor MRT3847_21399C [Mesembryanthemum crystallinum] 9 130 Mnom000372 +Sc.SIZ1 gb|AAB64849.1|Ydr409wp; CAI: 0.12 [Saccharomyces cerevisiae] 10 131 Mnom000412-Mnom000413 +Os.GF14h gb|ABA94733.1|14-3-3, putative, expressed [Oryza sativa (japonica cultivar-group)] 11 132 Mnom000444 +Lb. dbj|BAA12858.1|ferredoxin-dependent glutamate Ferredoxin- synthase [Leptolyngbya boryana] dependent glutamate synthase 12 133 Mnom000451 +Gm.G3083-like 2 gb|EAY72627.1|hypothetical protein OsI_000474 [Oryza sativa (indica cultivar- group)] 13 134 Mnom000471 +Sc.TPS-TPP ref|NP_010359.1|Phosphatase subunit of the trehalose-6-phosphate synthase/phosphatase complex, which synthesizes the storage carbohydrate trehalose; expression is induced by stress conditions and repressed by the Ras-cAMP pathway [Saccharomyces cerevisiae] 14 135 Mnom000480 +Zm.A1ZM027162_at_OSJ >gb|EAY93657.1|hypothetical protein NBa0020I02.17 OsI_014890 [Oryza sativa (indica cultivar- protein group)] 15 136 Mnom000481 +Zm.A1ZM003077_s_at_Os gb|EAY78482.1|hypothetical protein 10g0415800 OsI_032441 [Oryza sativa (indica protein cultivar-group)] 16 137 Mnom000485 +Zm.A1ZM058479_at_FAR1 gb|ABF95032.1|FAR1 family protein, expressed family [Oryza sativa (japonica cultivar-group)] protein, expressed 17 138 Mnom000486 +Zm.A1ZM016696_x_at_Hypothetical gb|EAY85998.1|hypothetical protein protein OsI_007231 [Oryza sativa (indica cultivar- OSJNBa0052M16.13 group)] 18 139 Mnom000490 +Zm.A1ZM057199_s_at_Peptide >gb|EAY89170.1|hypothetical protein transporter OsI_010403 [Oryza sativa (indica cultivar- PTR2, group)] putative, expressed 19 140 Mnom000491 +Zm.A1ZM060496_at_Expressed >gb|ABF94602.1|expressed protein [Oryza sativa protein (japonica cultivar-group)] 20 141 Mnom000494 −Zm.A1ZM012001_at_Putative ref|NP_001054859.1|Os05g0194900 [Oryza 6-phospho- sativa (japonica cultivar-group)] 1- fructokinase 21 142 Mnom000496 −Zm.A1ZM061308_at_Putative ref|NP_001058018.1|Os06g0604500 [Oryza small calcium sativa (japonica cultivar-group)] binding mitochondrial carrier 2 22 143 Mnom000497 −Zm.A1ZM073603_at_Putative gb|EAZ10866.1|hypothetical protein OsJ_000691 6-phospho- [Oryza sativa (japonica cultivar-group)] 1- fructokinase 23 144 Mnom000499 −Zm.A1ZM060016_at_Os02g0642600 gb|EAZ23967.1|hypothetical protein OsJ_007450 protein [Oryza sativa (japonica cultivar-group)] 24 145 Mnom000517 +Gh.MRT363517117P.2 gb|AAY67798.1|14-3-3 protein [Manihot esculenta] 25 146 Mnom000554 +Mt.CGPG2117 unnamed protein product [Vitis vinifera] 26 147 Mnom000570 +At.SIZ1 ATSIZ1/SIZ1; DNA binding [Arabidopsis thaliana] 27 148 Mnom000580 +Gh.SP26 alpha-crystalline heat shock protein [Gossypium arboreum] gb|ABI97959.1|alpha-crystalline heat shock protein [Gossypium arboreum] 28 149 Mnom000616-Mnom000617 +At.XDH1 ATXDH1 (XANTHINE DEHYDROGENASE 1); xanthine dehydrogenase [Arabidopsis thaliana] gb|AAO11781.1|xanthine dehydrogenase 1 [Arabidopsis thaliana] 29 150 Mnom000618-Mnom000619 +At.XDH2 xanthine dehydrogenase 2 [Arabidopsis thaliana] 30 151 Mnom000626-Mnom000627 +Zm.GA2ox Os01g0209700 [Oryza sativa (japonica cultivar- group)] dbj|BAA96178.1|putative GA 2-oxidase 5 [Oryza 8sativa (japonica cultivar-group)] dbj|BAF04278.1|Os01g0209700 [Oryza sativa (japonica cultivar-group)] 31 152 Mnom000628 −Zm.GA2ox Os01g0209700 [Oryza sativa (japonica cultivar- group)] dbj|BAA96178.1|putative GA 2-oxidase 5 [Oryza sativa (japonica cultivar-group)] dbj|BAF04278.1|Os01g0209700 [Oryza sativa (japonica cultivar-group)] 32 153 Mnom000636 +Zm.PHD finger hypothetical protein OsJ_019708 [Oryza sativa protein-like (japonica cultivar-group)] 33 154 Mnom000637 +Zm.04g0442000 Os04g0442000 [Oryza sativa (japonica cultivar- group)] sp|Q0JCZ4|ARFI_ORYSJ Auxin response factor 9 dbj|BAF14793.1| Os04g0442000 [Oryza sativa (japonica cultivar- group)] 34 155 Mnom000640 +Zm.P0415C01.9 unknown protein [Oryza sativa (japonica cultivar- group)] 35 156 Mnom000641 +Zm.5′-3′ Os03g0794800 [Oryza sativa (japonica cultivar- exoribonuclease, group)] gb|AAT76426.1|putative 5′-3′ putative, exonuclease [Oryza sativa (japonica cultivar- expressed group)] gb|ABF99326.1|5′-3′ exoribonuclease, putative, expressed [Oryza sativa (japonica cultivar-group)] dbj|BAF13456.1|Os03g0794800 [Oryza sativa (japonica cultivar-group)] 36 157 Mnom000643 +Zm.Putative Os06g0149900 [Oryza sativa (japonica cultivar- cysteine synthase group)] dbj|BAD69043.1|putative cysteine synthase [Oryza sativa (japonica cultivar-group)] dbj|BAF18734.1|Os06g0149900 [Oryza sativa (japonica cultivar-group)] 37 158 Mnom000645 +Zm.Putative Os10g0475000 [Oryza sativa (japonica cultivar- oxidase group)] gb|AAL31049.1|AC078893_12 putative oxidase [Oryza sativa] gb|ABB47786.2|GMC oxidoreductase family protein, expressed [Oryza sativa (japonica cultivar-group)] dbj|BAF26755.1| Os10g0475000 [Oryza sativa (japonica cultivar- group)] 38 159 Mnom000647 +Zm.Serine Serine carboxypeptidase family protein, carboxypeptidase expressed [Oryza sativa (japonica cultivar-group)] family protein, expressed 39 160 Mnom000682 +At.SQD2 SQD2 (SULFOQUINOVOSYLDIACYLGLYCEROL 2); UDP-sulfoquinovose:DAG sulfoquinovosyltransferase/transferase, transferring glycosyl groups [Arabidopsis thaliana] gb|AAM18913.1|AF454354_1 sulfolipid synthase [Arabidopsis thaliana] gb|AAO64198.1| unknown protein [Arabidopsis thaliana] 40 161 Mnom000690 +As.2-cys 2Cys-peroxiredoxin precursor [Brassica rapa] peroxiredoxin CTP fused to soy 2-cys peroxiredoxin enzyme domain 41 162 Mnom000107 +Zm.phyA2 phytochrome A2 [Zea mays] 42 163 Mnom000108 +Zm.phyA2 phytochrome A2 [Zea mays] 43 164 Mnom000109 +Zm.phyA2 phytochrome A2 [Zea mays] 44 165 Mnom000110 +Zm.phyB1 phytochrome B1 apoprotein; PhyB1 [Zea mays] 45 166 Mnom000111 +Zm.PhyB1 phytochrome B1 apoprotein; PhyB1 [Zea mays] 46 167 Mnom000112 +Zm.PhyB1 phytochrome B1 apoprotein; PhyB1 [Zea mays] 47 168 Mnom000113, +Zm.PhyA2 phytochrome A2 [Zea mays] Mnom000116 48 169 Mnom000114, +Zm.PhyB1 phytochrome B1 apoprotein; PhyB1 [Zea mays] Mnom000117 49 170 Mnom000115, +Zm.PhyB1 phytochrome B1 apoprotein; PhyB1 [Zea mays] Mnom000118 50 171 Mnom000119 +Zm.phyA2 phytochrome A2 [Zea mays] 51 172 Mnom000738 +Zm.RTCL RTCS-like protein [Zea mays] 52 173 Mnom000766-Mnom000767 +Cr.MoT1 molybdate transporter [Chlamydomonas reinhardtii] 53 174 Mnom000822-Mnom000823 −Zm.GW2 RING-type E3 ubiquitin ligase [Oryza sativa (indica cultivar-group)] 54 175 Mnom000857, +Zm.wilt1256 Os03g0291800 [Oryza sativa (japonica cultivar- Mnom000858, group)] Mnom000860 55 176 Mnom000873 +Th.CHIT33 chitinase [Hypocrea lixii] 56 177 Mnom000886 +Zm.SEC22 putative vesicle trafficking protein [Oryza sativa (japonica cultivar-group)] 57 178 Mnom000923-Mnom000924 +Zm.phyB1 phytochrome B1 apoprotein; PhyB1 [Zea mays] 58 179 Mnom000937 +Zm.NPH1 blue-light receptor phototropin 1 [Zea mays] gb|AAB88817.1|nonphototropic hypocotyl 1 [Zea mays] 59 180 Mnom000942, +Os.TOP6A1 putative topoisomerase 6 [Oryza sativa (japonica Mnom000955-Mnom000956 cultivar-group)] 60 181 Mnom000985 +Zm.A1ZM027162_at_OSJNBa0020I02.17 hypothetical protein OsI_014890 [Oryza sativa protein (indica cultivar-group)] 61 182 Mnom000986 +Zm.A1ZM003077_s_at_Os10g0415800 hypothetical protein OsI_032441 [Oryza sativa protein (indica cultivar-group)] 62 183 Mnom001055 +Zm.Serine Serine carboxypeptidase family protein, carboxypeptidase expressed [Oryza sativa (japonica cultivar-group)] family protein, expressed 63 184 Mnom001090-Mnom001092 +At.Glutamine GS2 (GLUTAMINE SYNTHETASE 2); synthetase glutamate-ammonia ligase [Arabidopsis thaliana] 64 185 Mnom001138 +Mt.UDPGPP UDP-glucose pyrophosphorylase [Amorpha fruticosa] 65 186 Mnom001140 +Mt.DNAJ unnamed protein product [Vitis vinifera] HEAT SHOCK N-TERMINAL DOMAIN- CONTAINING PROTEIN 66 187 Mnom001141 +Gh.DNAJ DnaJ-like protein [Glycine max] HEAT SHOCK N-TERMINAL DOMAIN- CONTAINING PROTEIN 67 188 Mnom001142 −Gm.CGPG1284 nicotinate phosphoribosyltransferase-like protein homologue [Medicago truncatula] 68 189 Mnom001146 +Zm.G1073-like 1 Os02g0713700 [Oryza sativa (japonica cultivar- group)] 69 190 Mnom001147 −Zm.G1073-like 1 Os02g0713700 [Oryza sativa (japonica cultivar- group)] 70 191 Mnom001160 +Zm.Znf1 zinc finger protein [Zea mays] 71 192 Mnom001162 +Zm.CSLD1 putative cellulose synthase [Oryza sativa (japonica cultivar-group)] 72 193 PHE0000597 +Ec.mannitol-1- mannitol-1-phosphate 5-dehydrogenase phosphate 5- [Escherichia coli CFT073] dehydrogenase gb|AAN82853.1|AE016768_271 Mannitol-1- phosphate 5-dehydrogenase [Escherichia coli CFT073] 73 194 PHE0001556 +Np.cphA COG0769: UDP-N-acetylmuramyl tripeptide synthase [Nostoc punctiforme PCC 73102] 74 195 PHE0001557 +Ss.cphA- hypothetical protein slr2002 [Synechocystis sp. 1652956 PCC 6803] sp|P73833|CPHA_SYNY3 Cyanophycin synthetase dbj|BAA17890.1|slr2002 [Synechocystis sp. PCC 6803] 75 196 PHE0001559 +Ss.cphB- hypothetical protein slr2001 [Synechocystis sp. 1652956 PCC 6803] sp|P73832|CPHB_SYNY3 Cyanophycinase dbj|BAA17889.1|slr2001 [Synechocystis sp. PCC 6803] 76 197 PHE0002862 +Os.Senescence Os05g0148700 [Oryza sativa (japonica cultivar- Associated group)] gb|AAS46022.1|PSAG protein; PSAG1 Protein [Oryza sativa (japonica cultivar-group)] gb|AAS46023.1|PSAG protein; OsPSAG [Oryza sativa (japonica cultivar-group)] gb|AAU44024.1| unknown protein [Oryza sativa (japonica cultivar- group)] dbj|BAF16565.1|Os05g0148700 [Oryza sativa (japonica cultivar-group)] 77 198 PHE0003348 +At.PIF4 PIF4 (PHYTOCHROME INTERACTING FACTOR 4); DNA binding/transcription factor [Arabidopsis thaliana] sp|Q8W2F3|PIF4_ARATH Phytochrome-interacting factor 4 (Basic helix- loop-helix protein 9) (bHLH 9) (AtbHLH009) (Short under red-light 2) gb|AAL55716.1|AF251694_1 putative transcription factor BHLH9 [Arabidopsis thaliana] emb|CAD29449.1|phytochrome interacting factor 4 [Arabidopsis thaliana] 78 199 PHE0003706 +Sc.DUR3 Dur3 79 200 PHE0003708 +Sp.DUR3 hypothetical protein SPBC23G7.13c [Schizosaccharomyces pombe 972h-] emb|CAA22629.1|urea transporter (predicted) [Schizosaccharomyces pombe] 80 201 PHE0004230 +Zm.E2F3 like Os02g0537500 [Oryza sativa (japonica cultivar- group)] dbj|BAD73815.1|putative E2F homolog [Oryza sativa (japonica cultivar-group)] dbj|BAF08963.1|Os02g0537500 [Oryza sativa (japonica cultivar-group)] 81 202 PHE0004820 +Zm. hypothetical protein OsJ_008481 [Oryza sativa MRT4577_21665C- (japonica cultivar-group)] like ZmPIF3- like family gene in branch 1 82 203 PHE0004853 −Zm.Wee1 Wee1-like protein [Zea mays] fragment 83 204 PHE0006017 +Gh.Na/H Na/H antiporter [Gossypium hirsutum] antiporter 84 205 PHE0006102 +Sc.CGPG8909 Constituent of 66S pre-ribosomal particles, SSF1_YEAST required for ribosomal Ribosome large subunit maturation; functionally redundant biogenesis with protein Ssf2p; member of the Brix family [Saccharomyces cerevisiae] 85 206 PHE0006378 +Sc.Rrp40p YOL142W [Saccharomyces cerevisiae] 86 207 PHE0006380 +Sc.Rrp46p Protein involved in rRNA processing; component of the exosome 3->5 exonuclease complex; Rrp46p [Saccharomyces cerevisiae] sp|P53256|RRP46_YEAST Exosome complex exonuclease RRP46 (Ribosomal RNA-processing protein 46) gb|EDN61685.1|3′-5′-exoribonuclease [Saccharomyces cerevisiae YJM789] 87 208 PHE0006496 −Zm.KNAT6 N/A New RNAi 88 209 PHE0006610 +Gm.Glutathione Phospholipid hydroperoxide glutathione Peroxidase peroxidase, chloroplast precursor (PHGPx) emb|CAA04142.1|phospholipid glutathione peroxidase [Pisum sativum] 89 210 PHE0006613 +Gm. Os03g0358100 [Oryza sativa (japonica cultivar- Glutathione group)] emb|CAC17628.1|putative phospholipid Peroxidase hydroperoxide glutathione peroxidase [Oryza sativa (japonica cultivar-group)] gb|ABF96054.1| glutathione peroxidase, putative, expressed [Oryza sativa (japonica cultivar-group)] dbj|BAF12059.1|Os03g0358100 [Oryza sativa (japonica cultivar-group)] gb|EAY90064.1| hypothetical protein OsI_011297 [Oryza sativa (indica cultivar-group)] gb|EAZ26982.1| hypothetical protein OsJ_010465 [Oryza sativa (japonica cultivar-group)] 90 211 PHE0006916 +Zm.A1ZM059972_at_Ribosome N/A associated membrane protein RAMP4 91 212 PHE0006917 +Zm.A1ZM057844_at_26S Os06g0633100 [Oryza sativa (japonica cultivar- protease group)] regulatory dbj|BAD37803.1|unknown protein [Oryza sativa subunit 7 (26S (japonica cultivar-group)] proteasome dbj|BAF20043.1|Os06g0633100 [Oryza sativa subunit 7) (japonica cultivar-group)] 92 213 PHE0007643 +At.CGPG7785_At4g22730 leucine-rich repeat transmembrane protein kinase, emb|CAB79228.1| putative [Arabidopsis thaliana] emb|CAA16558.1| leucine rich leucine rich repeat receptor kinase-like protein repeat [Arabidopsis thaliana] emb|CAB79228.1|leucine rich repeat receptor kinase-like protein [Arabidopsis thaliana] gb|AAR23717.1| At4g22730 [Arabidopsis thaliana] dbj|BAD44629.1|leucine rich repeat receptor kinase-like protein [Arabidopsis thaliana] 93 214 PHE0008287 +Zm.CGPG7793 Os05g0154900 [Oryza sativa (japonica cultivar- group)] gb|AAV32216.1|unknown protein [Oryza sativa (japonica cultivar-group)] dbj|BAF16600.1| Os05g0154900 [Oryza sativa (japonica cultivar- group)] 94 215 PHE0008407 +Ps.CGPG6515_succinate- succinate-semialdehyde dehydrogenase semialdehyde [Pseudomonas syringae pv. tomato str. DC3000] dehydrogenase gb|AAO53845.1|succinate-semialdehyde dehydrogenase [Pseudomonas syringae pv. tomato str. DC3000] 95 216 PHE0008411 +Zm.CGPG9112_hypothetical hypothetical protein OsJ_012412 [Oryza sativa protein (japonica cultivar-group)] 96 217 PHE0008541 +Zm.Ctp-Hb1 3-phosphoshikimate 1-carboxyvinyltransferase/ 5-enolpyruvylshikimate-3-phosphate/EPSP synthase [Arabidopsis thaliana] gb|AAB82633.1| 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase [Arabidopsis thaliana] gb|AAY25438.1| At2g45300 [Arabidopsis thaliana] dbj|BAE99170.1|5-enolpyruvylshikimate-3- phosphate (EPSP) synthase [Arabidopsis thaliana] 97 218 PHE0008602 +Xn.CGPG7423_glutamine glutamine synthetase [Photorhabdus luminescens synthetase subsp. laumondii TTO1] emb|CAE12532.1| (glutamate-- glutamine synthetase (glutamate--ammonia ammonia ligase) ligase) [Photorhabdus luminescens subsp. laumondii TTO1] 98 219 PHE0009339 +At.Luminal BIP (LUMINAL BINDING PROTEIN); ATP binding protein 2 binding [Arabidopsis thaliana] precursor sp|Q39043|BIP2_ARATH Luminal-binding protein 2 precursor (BiP2) (AtBP2) dbj|BAB08435.1|luminal binding protein [Arabidopsis thaliana] gb|AAO00752.1|luminal binding protein [Arabidopsis thaliana] gb|AAP37765.1|At5g42020 [Arabidopsis thaliana] 99 220 PHE0009341 +Os.Putative Os01g0307500 [Oryza sativa (japonica cultivar- high affinity group)] sp|Q3YAF4|HKT8_ORYSA Cation potassium transporter HKT8 (OsHKT8) transporter 2 dbj|BAB93392.1|putative high affinity potassium transporter 2 [Oryza sativa (japonica cultivar-group)] dbj|BAD88380.1|putative high affinity potassium transporter 2 [Oryza sativa (japonica cultivar-group)] dbj|BAF04762.1|Os01g0307500 [Oryza sativa (japonica cultivar-group)] 100 221 PHE0010195 +At.Arabidopsis oligopeptide transport (POT) family protein Peptide [Arabidopsis thaliana] transporter emb|CAB87717.1|putative oligopeptide transporter protein [Arabidopsis thaliana] 101 222 PHE0010352 +Gh.GhG481 transcription factor NF-Y CCAAT-binding-like protein-like [Solanum tuberosum] gb|ABB55391.1|transcription factor NF-Y CCAAT-binding-like protein-like [Solanum tuberosum] gb|ABB72810.1|transcription factor NF-Y, CCAAT-binding-like protein [Solanum tuberosum] 102 223 PHE0010354 +Gh.NFB2-1:1:1 transcription factor NF-Y CCAAT-binding-like protein-like [Solanum tuberosum] gb|ABB55391.1|transcription factor NF-Y CCAAT-binding-like protein-like [Solanum tuberosum] gb|ABB72810.1|transcription factor NF-Y, CCAAT-binding-like protein [Solanum tuberosum] 103 224 PHE0010646 +U.GOI- spermidine synthase [Cucumis sativus] CUCfi.FSPD1:1:1 104 225 PHE0010648 +Gh.G2041_Truncated HR38 (chromatin remodeling 38); ATP binding/ DNA binding/helicase [Arabidopsis thaliana] emb|CAB86450.1|putative protein [Arabidopsis thaliana 105 226 PHE0010649 +Gm.G2041_Truncated SNF2 domain-containing protein [Olimarabidopsis pumila] 106 227 PHE0010852 +Zm.Helix helix hypothetical protein OsI_028787 [Oryza sativa loop (indica cultivar-group)] protein_NC0035132: YLD 107 228 PHE0010956 +At.ENT1 ENT1, AT (EQUILIBRATIVE NUCLEOTIDE TRANSPORTER 1); nucleoside transporter [Arabidopsis thaliana] gb|AAL67041.1|unknown protein [Arabidopsis thaliana] gb|AAO22816.1|unknown protein [Arabidopsis thaliana] 108 229 PHE0011028 +U.GOI- N/A Nue1:1:1 109 230 PHE0011063 +U.GOI-Nue1- Non-symbiotic hemoglobin (Hbt) (ZEAmp Zm.Hb1RNA:1:1 GLB1) gb|AAF44664.1|AF236080_1 hemoglobin [Zea mays] gb|AAG01183.1|hemoglobin [Zea mays subsp. parviglumis] 110 231 PHE0011543 +Le.Tomato zinc finger (B-box type) family protein Homolog of [Arabidopsis thaliana] G1988 111 232 PHE0013202 +At.basic helix- PIL2 (PHYTOCHROME INTERACTING loop-helix FACTOR 3-LIKE 2); transcription (bHLH) protein factor [Arabidopsis thaliana] similar to PIF3- like bHLH protein 2 (PIL2) 112 233 PHE0013203 +At.sterile alpha sterile alpha motif (SAM) domain-containing motif (SAM) protein [Arabidopsis thaliana] domain- containing protein 113 234 PHE0013205 +At.DUF unknown protein [Arabidopsis thaliana] 114 235 PHE0013292 +At.ctpA C-terminal processing protease, putative [Arabidopsis thaliana] 115 236 PHE0013712 +Zm.PROPEP2 Os04g0638700 [Oryza sativa (japonica cultivar- group)] 116 237 PHE0013713 +Zm.PROPEP3 Os04g0638700 [Oryza sativa (japonica cultivar- group)] 117 238 PHE0013734 +At.AGL94 DNA binding/transcription factor [Arabidopsis thaliana] 118 239 TRG0000049 −Zm.NFB5 hypothetical protein OsI_019577 [Oryza sativa (indica cultivar-group)] 119 240 TRG0000291 −Zm.coumaryl AMP-binding enzyme family protein, expressed ligase [Oryza sativa (japonica cultivar-group)] 120 241 TRG0000332 −Gm.NFA9.3.1- unnamed protein product [Vitis vinifera] Soy homolog of AtG926 121 242 1071693 +Zm.PhyB phytochrome B2 apoprotein; PhyB2 [Zea mays] Selection Methods for Transgenic Plants with Enhanced Agronomic Trait

Within a population of transgenic plants each regenerated from a plant cell having a nucleus with recombinant DNA many plants that survive to fertile transgenic plants that produce seeds and progeny plants will not exhibit an enhanced agronomic trait. Selection from the population is necessary to identify one or more transgenic plant cells having a transgenic nucleus that can provide plants with the enhanced trait. Transgenic plants having enhanced traits are selected from populations of plants regenerated or derived from plant cells transformed as described herein by evaluating the plants in a variety of assays to detect an enhanced trait, e.g. enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil. These assays also may take many forms including, but not limited to, direct screening for the trait in a greenhouse or field trial or by screening for a surrogate trait. Such analyses can be directed to detecting changes in the chemical composition, biomass, physiological properties, morphology of the plant. Changes in chemical compositions such as nutritional composition of grain can be detected by analysis of the seed composition and content of protein, free amino acids, oil, free fatty acids, starch or tocopherols. Changes in biomass characteristics can be made on greenhouse or field grown plants and can include plant height, stem diameter, root and shoot dry weights; and, for corn plants, ear length and diameter. Changes in physiological properties can be identified by evaluating responses to stress conditions, for example assays using imposed stress conditions such as water deficit, nitrogen deficiency, cold growing conditions, pathogen or insect attack or light deficiency, or increased plant density. Changes in morphology can be measured by visual observation of tendency of a transformed plant with an enhanced agronomic trait to also appear to be a normal plant as compared to changes toward bushy, taller, thicker, narrower leaves, striped leaves, knotted trait, chlorosis, albino, anthocyanin production, or altered tassels, ears or roots. Other selection properties include days to pollen shed, days to silking, leaf extension rate, chlorophyll content, leaf temperature, stand, seedling vigor, internode length, plant height, leaf number, leaf area, tillering, brace roots, stay green, stalk lodging, root lodging, plant health, barreness/prolificacy, green snap, and pest resistance. In addition, phenotypic characteristics of harvested grain may be evaluated, including number of kernels per row on the ear, number of rows of kernels on the ear, kernel abortion, kernel weight, kernel size, kernel density and physical grain quality.

Assays for screening for a desired trait are readily designed by those practicing in the art. The following illustrates useful screening assays for corn traits using hybrid corn plants. The assays can be readily adapted for screening other plants such as canola, cotton and soybean either as hybrids or inbreds.

Transgenic corn plants having nitrogen use efficiency are identified by screening in fields with three levels of nitrogen (N) fertilizer being applied, e.g. low level (0 N), medium level (80 lb/ac) and high level (180 lb/ac). Plants with enhanced nitrogen use efficiency provide higher yield as compared to control plants.

Transgenic corn plants having enhanced yield are identified by screening using progeny of the transgenic plants over multiple locations with plants grown under optimal production management practices and maximum weed and pest control. A useful target for improved yield is a 5% to 10% increase in yield as compared to yield produced by plants grown from seed for a control plant. Selection methods may be applied in multiple and diverse geographic locations, for example up to 16 or more locations, over one or more planting seasons, for example at least two planting seasons, to statistically distinguish yield improvement from natural environmental effects.

Transgenic corn plants having enhanced water use efficiency are identified by screening plants in an assay where water is withheld for a period to induce stress followed by watering to revive the plants. For example, a useful selection process imposes 3 drought/re-water cycles on plants over a total period of 15 days after an initial stress free growth period of 11 days. Each cycle consists of 5 days, with no water being applied for the first four days and a water quenching on the 5th day of the cycle. The primary phenotypes analyzed by the selection method are the changes in plant growth rate as determined by height and biomass during a vegetative drought treatment.

Transgenic corn plants having enhanced cold tolerance are identified by screening plants in a cold germination assay and/or a cold tolerance field trial. In a cold germination assay trays of transgenic and control seeds are placed in a growth chamber at 9.7° C. for 24 days (no light). Seeds having higher germination rates as compared to the control are identified as having enhanced cold tolerance. In a cold tolerance field trial plants with enhanced cold tolerance are identified from field planting at an earlier date than conventional Spring planting for the field location. For example, seeds are planted into the ground around two weeks before local farmers begin to plant corn so that a significant cold stress is exerted onto the crop, named as cold treatment. Seeds also are planted under local optimal planting conditions such that the crop has little or no exposure to cold condition, named as normal treatment. At each location, seeds are planted under both cold and normal conditions preferably with multiple repetitions per treatment.

Transgenic corn plants having seeds with increased protein and/or oil levels are identified by analyzing progeny seed for protein and/or oil. Near-infrared transmittance spectrometry is a non-destructive, high-throughput method that is useful to determine the composition of a bulk seed sample for properties listed in table 2.

TABLE 2 Typical sample(s): Whole grain corn and soybean seeds Typical analytical range: Corn - moisture 5-15%, oil 5-20%, protein 5-30%, starch 50-75%, and density 1.0-1.3%. Soybean - moisture 5-15%, oil 15-25%, and protein 35-50%.

Although the plant cells and methods of this invention can be applied to any plant cell, plant, seed or pollen, e.g. any fruit, vegetable, grass, tree or ornamental plant, the various aspects of the invention are preferably applied to corn, soybean, cotton, canola, alfalfa, wheat, rice, sugarcane, and sugar beet plants. In many cases the invention is applied to corn plants that are inherently resistant to disease from the Mal de Rio Cuarto virus or the Puccina sorghi fungus or both.

The following examples are included to demonstrate aspects of the invention, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific aspects which are disclosed and still obtain a like or similar results without departing from the spirit and scope of the invention.

Example 1 Plant Expression Constructs

This example illustrates the construction of plasmids for transferring recombinant DNA into a plant cell nucleus that can be regenerated into transgenic plants.

A. Plant Expression Constructs for Corn Transformation

A base corn transformation vector pMON93039, as set forth in SEQ ID NO:17377, illustrated in Table 3 and FIG. 1, is fabricated for use in preparing recombinant DNA for Agrobacterium-mediated transformation into corn tissue.

TABLE 3 Coordinates of Function Name Annotation SEQ ID NO: 17377 Agrobacterium B-AGRtu.right border Agro right border sequence, 11364-11720 T-DNA transfer essential for transfer of T-DNA. Gene of interest E-Os.Act1 Upstream promoter region of the  19-775 expression rice actin 1 gene cassette E-CaMV.35S.2xA1- Duplicated35S A1-B3 domain  788-1120 B3 without TATA box P-Os.Act1 Promoter region of the rice actin 1 1125-1204 gene L-Ta.Lhcb1 5′ untranslated leader of wheat 1210-1270 major chlorophyll a/b binding protein I-Os.Act1 First intron and flanking UTR exon 1287-1766 sequences from the rice actin 1 gene T-St.Pis4 3′ non-translated region of the 1838-2780 potato proteinase inhibitor II gene which functions to direct polyadenylation of the mRNA Plant selectable P-Os.Act1 Promoter from the rice actin 1 gene 2830-3670 marker L-Os.Act1 First exon of the rice actin 1 gene 3671-3750 expression I-Os.Act1 First intron and flanking UTR exon 3751-4228 cassette sequences from the rice actin 1 gene TS-At.ShkG-CTP2 Transit peptide region of 4238-4465 Arabidopsis EPSPS CR-AGRtu.aroA- Coding region for bacterial strain 4466-5833 CP4.nat CP4 native aroA gene. T-AGRtu.nos A 3′ non-translated region of the 5849-6101 nopaline synthase gene of Agrobacterium tumefaciens Ti plasmid which functions to direct polyadenylation of the mRNA. Agrobacterium B-AGRtu.left border Agro left border sequence, essential 6168-6609 T-DNA transfer for transfer of T-DNA. Maintenance in OR-Ec.oriV-RK2 The vegetative origin of replication 6696-7092 E. coli from plasmid RK2. CR-Ec.rop Coding region for repressor of 8601-8792 primer from the ColE1 plasmid. Expression of this gene product interferes with primer binding at the origin of replication, keeping plasmid copy number low. OR-Ec.ori-ColE1 The minimal origin of replication 9220-9808 from the E. coli plasmid ColE1. P-Ec.aadA-SPC/STR Promoter for Tn7 10339-10380 adenylyltransferase (AAD(3″)) CR-Ec.aadA- Coding region for Tn7 10381-11169 SPC/STR adenylyltransferase (AAD(3″)) conferring spectinomycin and streptomycin resistance. T-Ec.aadA-SPC/STR 3′ UTR from the Tn7 11170-11227 adenylyltransferase (AAD(3″)) gene of E. coli.

To construct transformation vectors for expressing a protein identified in Table 1, primers for PCR amplification of the protein coding nucleotides are designed at or near the start and stop codons of the coding sequence, in order to eliminate most of the 5′ and 3′ untranslated regions. The protein coding nucleotides are inserted into the base vector in the gene of interest expression cassette at an insertion site, i.e. between the intron element (coordinates 1287-1766) and the polyadenylation element (coordinates 1838-2780).

To construct transformation vectors for suppressing a protein identified in Table 1, the amplified protein coding nucleotides are assembled in a sense and antisense arrangement and inserted into the base vector at the insertion site in the gene of interest expression cassette to provide transcribed RNA that will form a double-stranded RNA for RNA interference suppression of the protein. More specifically, the sense and anti-sense DNA is derived from an endogenous corn gene that expresses a corn protein with an amino acid sequence of SEQ ID NO: 141-144, 152, 174, 190, 203, 208, or 239-240 or the corn homolog of SEQ ID NOs:122, 188, or 241.

B. Plant Expression Constructs for Soy and Canola Transformation

Vectors for use in transformation of soybean and canola tissue are prepared having the elements of expression vector pMON82053 (SEQ ID NO: 17378) as shown in Table 4 below and FIG. 2.

TABLE 4 Coordinates of Function Name Annotation SEQ ID NO: 17378 Agrobacterium T- B-AGRtu.left border Agro left border sequence, essential for 6144-6585 DNA transfer transfer of T-DNA. Plant selectable P-At.Act7 Promoter from the Arabidopsis actin 7 gene 6624-7861 marker expression L-At.Act7 5′UTR of Arabidopsis Act7 gene cassette I-At.Act7 Intron from the Arabidopsis actin7 gene TS-At.ShkG-CTP2 Transit peptide region of Arabidopsis 7864-8091 EPSPS CR-AGRtu.aroA- Synthetic CP4 coding region with dicot 8092-9459 CP4.nno_At preferred codon usage. T-AGRtu.nos A 3′ non-translated region of the nopaline 9466-9718 synthase gene of Agrobacterium tumefaciens Ti plasmid which functions to direct polyadenylation of the mRNA. Gene of interest P-CaMV.35S-enh Promoter for 35S RNA from CaMV  1-613 expression cassette containing a duplication of the −90 to −350 region. T-Gb.E6-3b 3′ untranslated region from the fiber protein  688-1002 E6 gene of sea-island cotton. Agrobacterium T- B-AGRtu.right Agro right border sequence, essential for 1033-1389 DNA transfer border transfer of T-DNA. Maintenance in E. coli OR-Ec.oriV-RK2 The vegetative origin of replication from 5661-6057 plasmid RK2. CR-Ec.rop Coding region for repressor of primer from 3961-4152 the ColE1 plasmid. Expression of this gene product interferes with primer binding at the origin of replication, keeping plasmid copy number low. OR-Ec.ori-ColE1 The minimal origin of replication from the 2945-3533 E. coli plasmid ColE1. P-Ec.aadA-SPC/STR Promoter for Tn7 adenylyltransferase 2373-2414 (AAD(3″)) CR-Ec.aadA- Coding region for Tn7 adenylyltransferase 1584-2372 SPC/STR (AAD(3″)) conferring spectinomycin and streptomycin resistance. T-Ec.aadA-SPC/STR 3′ UTR from the Tn7 adenylyltransferase 1526-1583 (AAD(3″)) gene of E. coli.

To construct transformation vectors for expressing a protein identified in Table 1, primers for PCR amplification of the protein coding nucleotides are designed at or near the start and stop codons of the coding sequence, in order to eliminate most of the 5′ and 3′ untranslated regions. The protein coding nucleotides are inserted into the base vector in the gene of interest expression cassette at an insertion site, i.e. between the promoter element (coordinates 1-613) and the polyadenylation element (coordinates 688-1002).

To construct transformation vectors for suppressing a protein identified in Table 1, the amplified protein coding nucleotides are assembled in a sense and antisense arrangement and inserted into the base vector at the insertion site in the gene of interest expression cassette to provide transcribed RNA that will form a double-stranded RNA for RNA interference suppression of the protein. More specifically, for soybean the sense and anti-sense DNA is derived from an endogenous soybean gene that expresses a soybean protein with an amino acid sequence of SEQ ID NOs: 122, 188, or 241 or the soybean homolog of SEQ ID NOs: 141-144, 152, 174, 190, 203, 208, or 239-240, and for canola the sense and anti-sense DNA is derived from an endogenous canola gene that encodes the canola homolog of SEQ ID NOs: 122, 141-144, 152, 174, 188, 190, 203, 208, or 239-241.

C. Cotton Transformation Vector

Plasmids for use in transformation of cotton tissue are prepared with elements of expression vector pMON99053 (SEQ ID NO: 17379) as shown in Table 5 below and FIG. 3.

TABLE 5 Coordinates of SEQ ID NO: Function Name Annotation 17379 Agrobacterium T- B-AGRtu.right border Agro right border sequence, essential for  1-357 DNA transfer transfer of T-DNA. Gene of interest Exp-CaMV.35S- Enhanced version of the 35S RNA promoter  388-1091 expression cassette enh + Ph.DnaK from CaMV plus the petunia hsp70 5′ untranslated region T-Ps.RbcS2-E9 The 3′ non-translated region of the pea RbcS2 1165-1797 gene which functions to direct polyadenylation of the mRNA. Plant selectable Exp-CaMV.35S Promoter and 5′ untranslated region from the 1828-2151 marker expression 35S RNA of CaMV cassette CR-Ec.nptII-Tn5 Coding region for neomycin 2185-2979 phosphotransferase gene from transposon Tn5 which confers resistance to neomycin and kanamycin. T-AGRtu.nos A 3′ non-translated region of the nopaline 3011-3263 synthase gene of Agrobacterium tumefaciens Ti plasmid which functions to direct polyadenylation of the mRNA. Agrobacterium T- B-AGRtu.left border Agro left border sequence, essential for 3309-3750 DNA transfer transfer of T-DNA. Maintenance in E. coli OR-Ec.oriV-RK2 The vegetative origin of replication from 3837-4233 plasmid RK2. CR-Ec.rop Coding region for repressor of primer from 5742-5933 the ColE1 plasmid. Expression of this gene product interferes with primer binding at the origin of replication, keeping plasmid copy number low. OR-Ec.ori-ColE1 The minimal origin of replication from the E. coli 6361-6949 plasmid ColE1. P-Ec.aadA-SPC/STR Promoter for Tn7 adenylyltransferase 7480-7521 (AAD(3″)) CR-Ec.aadA-SPC/STR Coding region for Tn7 adenylyltransferase 7522-8310 (AAD(3″)) conferring spectinomycin and streptomycin resistance. T-Ec.aadA-SPC/STR 3′ UTR from the Tn7 adenylyltransferase 8311-8368 (AAD(3″)) gene of E. coli.

To construct transformation vectors for expressing a protein identified in Table 1, primers for PCR amplification of the protein coding nucleotides are designed at or near the start and stop codons of the coding sequence, in order to eliminate most of the 5′ and 3′ untranslated regions. The protein coding nucleotides are inserted into the base vector in the gene of interest expression cassette at an insertion site, i.e. between the promoter element (coordinates 388-1091) and the polyadenylation element (coordinates 1165-1797).

To construct transformation vectors for suppressing a protein identified in Table 1, the amplified protein coding nucleotides are assembled in a sense and antisense arrangement and inserted into the base vector at the insertion site in the gene of interest expression cassette to provide transcribed RNA that will form a double-stranded RNA for RNA interference suppression of the protein. More specifically, the sense and anti-sense DNA is derived from an endogenous cotton gene that encodes the cotton homolog of SEQ ID NO: 122, 141-144, 152, 174, 188, 190, 203, 208, or 239-241.

D. Plant Expression Constructs for Gene Stacking in Corn.

A base corn transformation vector pMON96782, as set forth in SEQ ID NO: 17380, illustrated in Table 6 and FIG. 4, is fabricated for use in preparing recombinant DNA for Agrobacterium-mediated transformation into corn tissue.

TABLE 6 Coordinates of Function Name Annotation SEQ ID NO: 17380 Agrobacterium B-AGRtu.right border Agro right border sequence,  1-357 T-DNA transfer essential for transfer of T-DNA. Gene of interest P-Os.Act1 Promoter region of the rice actin 1  403-1243 expression gene cassette 1 L-Os.Act1 5′ untranslated leader of rice actin 1 1244-1323 gene I-Os.Act1 First intron and flanking UTR exon 1324-1801 sequences from the rice actin 1 gene T-Ta.Hsp17 3′ un-translated region of wheat low 1834-2043 molecular weight heat shock protein gene Gene of interest E-Os.Act1 Upstream Promoter region of rice 2136-2892 expression actin 1 cassette 2 E-CaMV.35S.2xA1- 35S A1-B3 Domain 2905-2937 B3 P-Os.Act1 Promoter from rice actin gene 3242-3321 L-Ta.Lhcb1 5′ untranslated leader from wheat 3327-3387 chlorophyll protein I-Os.Act1 Intron and 5′ untranslated region 3404-3883 from rice actin 1 gene T-AGRtu.tr7 3′ untranslated region from 3918-4425 “transcript 7” of Agrobacterium Plant selectable P.Os.TubA Promoter of alpha-tubulin gene of 4452-5650 marker rice expression L.Os.TubA 5′ untranslated region of an alpha 5651-5736 cassette tubulin from rice I.Os.TubA Intron 1 of an alpha tubulin from 5737-6632 rice. Ts.Ta.waxy.nno_Zm Chloroplast transit peptide from 6637-6846 wheat starch synthase Cr.AGRtu.aroA- CP4 EPSPS gene 6847-8214 CP4.nno_Zm T.Os.TubA 3′ untranslated region of alpha 8219-8800 tubulin from rice Agrobacterium B-AGRtu.left border Left border sequence for T-DNA 8828-9269 T-DNA transfer transfer Maintenance in OR-Ec.oriV-RK2 Origin of replication from the E. coli 9356-9752 E. coli plasmid RK2. Cr-Ec.rop Coding region for repressor of 11261-11452 primer from ColE1 plasmid OR-Ec.ori-ColE1 Minimum origin of replication from 11880-12468 E. coli colE1 plasmid. P-Ec.aadA-SPC/STR Promoter for Tn7 12999-13040 adenylyltransferase gene CR-Ec.aadA- Coding region for Tn7 13041-13829 SPC/STR adenylyltransferase gene T-Ec.aadA-SPC/STR 3′ untranslated region from Tn7 13830-13887 adenylyltransferase gene

Primers for PCR amplification of protein coding nucleotides of the genes of interest are designed at or near the start and stop codons of the coding sequence, in order to eliminate most of the 5′ and 3′ untranslated regions. Protein coding regions of genes encoding a first and second protein of interest are amplified. The amplified region from the first gene of interest is cloned between nucleotides 1801 and 1834 of the base vector and the amplified region from the second gene of interest is cloned between nucleotides 3883 and 3918 of the base vector.

Example 2 Corn Transformation

This example illustrates transformation methods useful in producing a transgenic nucleus in a corn plant cell, and the plants, seeds and pollen produced from a transgenic cell with such a nucleus having an enhanced trait, i.e. enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil. A plasmid vector is prepared by cloning the DNA of SEQ ID NO:1 into the gene of interest expression cassette in the base vector for use in corn transformation of corn tissue provided in Example 1, Table 3.

For Agrobacterium-mediated transformation of corn embryo cells corn plants of a readily transformable line are grown in the greenhouse and ears are harvested when the embryos are 1.5 to 2.0 mm in length. Ears are surface sterilized by spraying or soaking the ears in 80% ethanol, followed by air drying. Immature embryos are isolated from individual kernels on surface sterilized ears. Prior to inoculation of maize cells, Agrobacterium cells are grown overnight at room temperature. Immature maize embryo cells are inoculated with Agrobacterium shortly after excision, and incubated at room temperature with Agrobacterium for 5-20 minutes. Immature embryo plant cells are then co-cultured with Agrobacterium for 1 to 3 days at 23° C. in the dark. Co-cultured embryos are transferred to selection media and cultured for approximately two weeks to allow embryogenic callus to develop. Embryogenic callus is transferred to culture medium containing 100 mg/L paromomycin and subcultured at about two week intervals. Transformed plant cells are recovered 6 to 8 weeks after initiation of selection.

For Agrobacterium-mediated transformation of maize callus immature embryos are cultured for approximately 8-21 days after excision to allow callus to develop. Callus is then incubated for about 30 minutes at room temperature with the Agrobacterium suspension, followed by removal of the liquid by aspiration. The callus and Agrobacterium are co-cultured without selection for 3-6 days followed by selection on paromomycin for approximately 6 weeks, with biweekly transfers to fresh media. Paromomycin resistant calli are identified about 6-8 weeks after initiation of selection.

To regenerate transgenic corn plants a callus of transgenic plant cells resulting from transformation and selection is placed on media to initiate shoot development into plantlets which are transferred to potting soil for initial growth in a growth chamber at 26° C. followed by a mist bench before transplanting to 5 inch pots where plants are grown to maturity. The regenerated plants are self-fertilized and seed is harvested for use in one or more methods to select seeds, seedlings or progeny second generation transgenic plants (R2 plants) or hybrids, e.g. by selecting transgenic plants exhibiting an enhanced trait as compared to a control plant.

The above process is repeated to produce multiple events of transgenic corn plant cells that are transformed with recombinant DNA from each of the genes identified in Table 1. Events are designed to produce in the transgenic cells one of the proteins identified in Table 1, except the proteins of SEQ ID NOs: 141-144, 152, 174, 190, 203, 208, and 239-240, and the corn homolog of SEQ ID NOs: 122, 188, and 241 which are suppressed. Progeny transgenic plants and seed of the transformed plant cells are screened for enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil. From each group of multiple events of transgenic plants with a specific recombinant DNA from Table 1 the event that produces the greatest enhancement in yield, water use efficiency, nitrogen use efficiency, enhanced cold tolerance, enhanced seed protein and enhanced seed oil is identified and progeny seed is selected for commercial development.

Example 3 Soybean Transformation

This example illustrates plant transformation useful in producing a transgenic nucleus in a soybean plant cell, and the plants, seeds and pollen produced from a transgenic cell with such a nucleus having an enhanced trait, i.e. enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil.

For Agrobacterium mediated transformation, soybean seeds are imbided overnight and the meristem explants excised. The explants are placed in a wounding vessel. Soybean explants and induced Agrobacterium cells from a strain containing plasmid DNA with the gene of interest cassette and a plant selectable marker cassette are mixed no later than 14 hours from the time of initiation of seed imbibition, and wounded using sonication. Following wounding, explants are placed in co-culture for 2-5 days at which point they are transferred to selection media for 6-8 weeks to allow selection and growth of transgenic shoots. Resistant shoots are harvested approximately 6-8 weeks and placed into selective rooting media for 2-3 weeks. Shoots producing roots are transferred to the greenhouse and potted in soil. Shoots that remain healthy on selection, but do not produce roots are transferred to non-selective rooting media for an additional two weeks. Roots from any shoots that produce roots off selection are tested for expression of the plant selectable marker before they are transferred to the greenhouse and potted in soil.

The above process is repeated to produce multiple events of transgenic soybean plant cells that are transformed with recombinant DNA from each of the genes identified in Table 1. Events are designed to produce in the transgenic cells one of the proteins identified in Table 1, except the proteins of SEQ ID NOs: 122, 188, and 241 and the soybean homologs of SEQ ID NOs: 141-144, 152, 174, 190, 203, 208, and 239-240, which are suppressed. Progeny transgenic plants and seed of the transformed plant cells are screened for enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced seed protein and enhanced seed oil. From each group of multiple events of transgenic plants with a specific recombinant DNA from Table 1 the event that produces the greatest enhancement in yield, water use efficiency, nitrogen use efficiency, enhanced cold tolerance, enhanced seed protein and enhanced seed oil is identified and progeny seed is selected for commercial development.

Example 4 Cotton Transgenic Plants with Enhanced Agronomic Traits

This example illustrates plant transformation useful in producing a transgenic nucleus in a cotton plant cell, and the plants, seeds and pollen produced from a transgenic cell with such a nucleus having an enhanced trait, i.e. enhanced water use efficiency, increased yield, enhanced nitrogen use efficiency and enhanced seed oil.

Transgenic cotton plants containing each recombinant DNA having a sequence of SEQ ID NO: 1 through SEQ ID NO: 121 are obtained by transforming with recombinant DNA from each of the genes identified in Table 1 using Agrobacterium-mediated transformation. The above process is repeated to produce multiple events of transgenic cotton plant cells that are transformed with recombinant DNA from each of the genes identified in Table 1. Events are designed to produce in the transgenic cells one of the proteins identified in Table 1, except the cotton homologs of the proteins of SEQ ID NOs: 122, 141-144, 152, 174, 188, 190, 203, 208, and 239-241 which are suppressed.

From each group of multiple events of transgenic plants with a specific recombinant DNA from Table 1 the event that produces the greatest enhancement in yield, water use efficiency, nitrogen use efficiency, enhanced cold tolerance, enhanced seed protein and enhanced seed oil is identified and progeny seed is selected for commercial development.

Progeny transgenic plants are selected from a population of transgenic cotton events under specified growing conditions and are compared with control cotton plants. Control cotton plants are substantially the same cotton genotype but without the recombinant DNA, for example, either a parental cotton plant of the same genotype that was not transformed with the identical recombinant DNA or a negative isoline of the transformed plant. Additionally, a commercial cotton cultivar adapted to the geographical region and cultivation conditions, i.e. cotton variety ST474, cotton variety FM 958, and cotton variety Siokra L-23, are used to compare the relative performance of the transgenic cotton plants containing the recombinant DNA.

Transgenic cotton plants with enhanced yield and water use efficiency are identified by growing under variable water conditions. Specific conditions for cotton include growing a first set of transgenic and control plants under “wet” conditions, i.e. irrigated in the range of 85 to 100 percent of evapotranspiration to provide leaf water potential of −14 to −18 bars, and growing a second set of transgenic and control plants under “dry” conditions, i.e. irrigated in the range of 40 to 60 percent of evapotranspiration to provide a leaf water potential of −21 to −25 bars. Pest control, such as weed and insect control is applied equally to both wet and dry treatments as needed. Data gathered during the trial includes weather records throughout the growing season including detailed records of rainfall; soil characterization information; any herbicide or insecticide applications; any gross agronomic differences observed such as leaf morphology, branching habit, leaf color, time to flowering, and fruiting pattern; plant height at various points during the trial; stand density; node and fruit number including node above white flower and node above crack boll measurements; and visual wilt scoring. Cotton boll samples are taken and analyzed for lint fraction and fiber quality. The cotton is harvested at the normal harvest timeframe for the trial area. Enhanced water use efficiency is indicated by increased yield, improved relative water content, enhanced leaf water potential, increased biomass, enhanced leaf extension rates, and improved fiber parameters.

Example 5 Canola Transformation

This example illustrates plant transformation useful in producing the transgenic canola plants of this invention and the production and identification of transgenic seed for transgenic canola having enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil.

Tissues from in vitro grown canola seedlings are prepared and inoculated with overnight-grown Agrobacterium cells containing plasmid DNA with the gene of interest cassette and a plant selectable marker cassette. Following co-cultivation with Agrobacterium, the infected tissues are allowed to grow on selection to promote growth of transgenic shoots, followed by growth of roots from the transgenic shoots. The selected plantlets are then transferred to the greenhouse and potted in soil. Molecular characterizations are performed to confirm the presence of the gene of interest, and its expression in transgenic plants and progenies. Progeny transgenic plants are selected from a population of transgenic canola events under specified growing conditions and are compared with control canola plants. Control canola plants are substantially the same canola genotype but without the recombinant DNA, for example, either a parental canola plant of the same genotype that is not transformed with the identical recombinant DNA or a negative isoline of the transformed plant.

Transgenic canola plant cells are transformed with each of the recombinant DNA identified in Table 1. The above process is repeated to produce multiple events of transgenic canola plant cells that are transformed with recombinant DNA from each of the genes identified in Table 1. Events are designed to produce in the transgenic cells one of the proteins identified in Table 1, except the canola homologs of the proteins of SEQ ID NOs: 122, 141-144, 152, 174, 188, 190, 203, 208, and 239-241 which are suppressed. Progeny transgenic plants and seed of the transformed plant cells are screened for enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced seed protein and enhanced seed oil. From each group of multiple events of transgenic plants with a specific recombinant DNA from Table 1 the event that produces the greatest enhancement in yield, water use efficiency, nitrogen use efficiency, enhanced cold tolerance, enhanced seed protein and enhanced seed oil is identified and progeny seed is selected for commercial development.

Example 6 Homolog Identification

This example illustrates the identification of homologs of proteins encoded by the DNA identified in Table 1 which is used to provide transgenic seed and plants having enhanced agronomic traits. From the sequence of the homologs, homologous DNA sequence can be identified for preparing additional transgenic seeds and plants of this invention with enhanced agronomic traits.

An “All Protein Database” was constructed of known protein sequences using a proprietary sequence database and the National Center for Biotechnology Information (NCBI) non-redundant amino acid database (nr.aa). For each organism from which a polynucleotide sequence provided herein was obtained, an “Organism Protein Database” was constructed of known protein sequences of the organism; it is a subset of the All Protein Database based on the NCBI taxonomy ID for the organism.

The All Protein Database was queried using amino acid sequences provided herein as SEQ ID NO: 122 through SEQ ID NO: 242 using NCBI “blastp” program with E-value cutoff of 1e-8. Up to 1000 top hits were kept, and separated by organism names. For each organism other than that of the query sequence, a list was kept for hits from the query organism itself with a more significant E-value than the best hit of the organism. The list contains likely duplicated genes of the polynucleotides provided herein, and is referred to as the Core List. Another list was kept for all the hits from each organism, sorted by E-value, and referred to as the Hit List.

The Organism Protein Database was queried using polypeptide sequences provided herein as SEQ ID NO: 122 through SEQ ID NO: 242 using NCBI “blastp” program with E-value cutoff of 1e-4. Up to 1000 top hits were kept. A BLAST searchable database was constructed based on these hits, and is referred to as “SubDB”. SubDB is queried with each sequence in the Hit List using NCBI “blastp” program with E-value cutoff of 1e-8. The hit with the best E-value was compared with the Core List from the corresponding organism. The hit is deemed a likely ortholog if it belongs to the Core List, otherwise it is deemed not a likely ortholog and there is no further search of sequences in the Hit List for the same organism. Homologs from a large number of distinct organisms were identified and are reported below in table 7 with the SEQ ID NO of the original query sequence and the identified homologs as [SEQ ID NO]: [Homolog SEQ ID NOs].

TABLE 7 Protein Sequences and their Homologs 122: 244 270 291 322 334 352 364 391 406 427 431 433 456 459 468 470 482 541 570 577 593 599 640 651 654 672 676 714 748 761 781 782 789 796 798 799 831 833 857 864 885 891 898 902 904 911 933 955 960 970 985 990 1005 1026 1033 1034 1044 1056 1065 1069 1075 1127 1133 1136 123: 272 315 328 345 360 414 430 447 460 464 476 561 565 566 591 633 636 650 662 699 703 708 710 725 755 765 819 826 828 836 852 866 883 909 923 932 996 1040 1111 1126 1128 1129 1130 1140 1144 1171 124: 269 320 321 324 336 358 370 377 384 387 408 420 429 440 445 465 477 488 502 528 586 598 600 606 628 663 667 697 698 702 706 745 791 805 806 859 913 931 934 952 953 1046 1070 1097 1099 1102 1113 1118 1120 1122 1147 1151 1157 1161 1162 1168 125: 249 258 263 271 286 335 337 338 344 350 351 375 390 402 405 411 426 438 439 449 454 458 493 499 517 562 563 571 572 573 589 594 603 624 627 631 718 720 739 768 772 776 784 788 793 837 886 889 896 907 925 939 943 944 950 957 963 964 969 976 1002 1008 1010 1014 1035 1039 1074 1083 1098 1104 1137 1146 1170 1177 1180 126: 243 245 246 247 248 250 252 253 254 255 256 257 259 260 261 262 264 265 266 267 268 273 274 276 277 278 279 280 282 284 285 287 288 290 292 293 295 296 297 298 300 301 302 303 304 305 306 307 308 309 310 311 314 316 317 318 319 323 325 326 327 330 331 332 333 339 340 341 342 343 346 348 349 354 355 356 357 359 362 363 365 368 369 371 372 373 374 376 378 379 380 381 382 383 388 389 392 393 394 395 396 398 399 400 403 404 407 409 410 413 415 417 418 419 421 422 423 425 432 435 436 441 442 443 446 448 451 452 453 455 461 462 463 466 467 469 471 472 473 474 475 479 481 483 484 485 486 487 491 492 495 496 497 498 500 501 504 505 506 507 508 509 510 511 512 513 514 515 516 518 522 523 524 525 526 527 529 531 532 533 534 536 537 538 539 540 542 543 544 545 546 547 548 549 550 551 553 554 555 556 557 558 559 564 567 568 569 575 576 579 580 581 582 583 584 585 587 588 592 595 596 597 602 604 607 608 610 611 613 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1071 1132 1134 129: 1188 1200 1204 1214 1232 1237 1273 1287 1291 1296 1313 1327 1334 1337 1338 1344 1369 1383 1418 1436 1444 1446 1451 1462 1473 1510 1536 1618 1633 1642 1708 1736 1750 1753 1767 1793 1798 1804 1864 1897 1902 1905 1931 1957 1958 1963 1966 1973 1986 2001 2022 2032 2041 2043 2095 2125 2146 2150 2163 2204 2211 2214 2252 2261 2305 2314 2315 2319 2328 2347 2348 2350 2352 2353 2354 2363 2408 2440 2445 2473 2480 2487 2511 2528 2566 2589 2610 2650 2654 2661 2685 2718 2737 2766 2775 2782 2802 2817 2844 2851 2863 2880 2895 2897 2907 2930 2941 2946 130: 1215 1219 1222 1270 1349 1358 1360 1406 1474 1530 1560 1562 1570 1597 1612 1650 1669 1691 1723 1727 1742 1745 1797 1813 1856 1873 1876 1919 1968 1999 2046 2165 2215 2281 2296 2325 2330 2331 2344 2438 2476 2517 2522 2537 2578 2658 2674 2683 2754 2780 2811 2830 2892 2913 2928 131: 1182 1206 1208 1243 1253 1278 1285 1286 1292 1299 1301 1345 1412 1489 1492 1503 1582 1588 1620 1634 1636 1639 1641 1653 1678 1712 1754 1807 1818 1848 1926 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2454 2478 2524 2626 2805 2829 2853 142: 1293 1434 1563 1610 1632 1657 1699 1724 1853 1882 1887 2003 2031 2162 2377 2439 2446 2451 2483 2565 2621 2645 2677 2679 2859 2876 2904 2924 143: 1252 1254 1266 1464 1483 1675 1775 1788 1978 1985 2058 2149 2254 2279 2346 2383 2454 2478 2524 2552 2626 2734 2805 2829 2853 2945 145: 1206 1213 1229 1241 1245 1253 1278 1285 1286 1290 1292 1294 1299 1301 1309 1333 1345 1364 1368 1376 1384 1388 1403 1405 1408 1424 1433 1443 1450 1485 1488 1503 1504 1517 1533 1543 1544 1556 1585 1620 1634 1636 1639 1641 1655 1677 1678 1695 1710 1712 1715 1743 1751 1754 1773 1782 1785 1805 1807 1810 1812 1818 1831 1851 1852 1862 1863 1899 1908 1926 1939 1952 1961 1972 1991 1996 2000 2002 2012 2019 2044 2054 2068 2072 2076 2083 2093 2096 2103 2154 2172 2179 2183 2209 2220 2225 2226 2227 2229 2230 2231 2244 2245 2247 2248 2258 2259 2260 2291 2298 2356 2358 2370 2389 2398 2401 2407 2411 2413 2418 2424 2448 2452 2456 2457 2459 2464 2466 2477 2490 2513 2516 2526 2532 2533 2547 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15496 15499 15504 15510 15515 15518 15521 15528 15561 15569 15571 15574 15583 15605 15623 15625 15635 15638 15681 15699 15708 15715 15792 15793 15797 15817 15821 15838 15841 15850 15856 15861 15873 15882 15883 15889 15891 15900 15931 15937 15938 15963 15964 15971 15974 15977 16007 16019 16024 16043 16081 16109 16118 16119 16191 16224 16229 16232 16234 16235 16261 16287 16291 16297 16299 16309 16343 16344 16353 16390 16404 16411 16414 16486 16507 16557 16563 16564 16585 16599 16602 16612 16624 16643 16649 16658 16670 16672 16673 16681 16696 16709 16716 16729 16730 16734 16739 16748 16759 16763 16776 16789 16802 16809 16825 16842 16859 16860 16901 16903 16909 16961 16974 16983 16991 16998 17000 17003 17005 17007 17018 17022 17027 17030 17066 17067 17078 17092 17100 17101 17113 17128 17149 17175 17179 17182 17191 17194 17203 17204 17207 17215 17232 17262 17265 17268 17271 17280 17283 17285 17306 17313 17376 226: 10662 10775 10801 10804 10952 11117 11193 11214 11231 11649 11651 11676 11678 11679 11814 12163 12228 12400 12800 13106 13171 13340 13396 13508 13717 13983 14014 14099 14132 14190 14277 14380 14472 14753 14837 14843 15189 15420 15490 15605 15635 16624 16649 16730 16763 16909 17027 17113 17191 17207 17280 227: 10950 11017 11107 11198 11210 11304 11517 11566 11759 11777 11846 11900 11929 11964 12165 12176 12257 12351 12629 12826 12987 13113 13472 13715 13771 13865 13921 13935 14082 14171 14280 14288 14311 14643 14769 14927 15245 15360 15440 15684 15732 16140 16150 16207 16275 16496 16611 16650 16979 228: 10838 10889 11126 11393 11561 11858 12024 13367 13608 13609 13656 13827 13864 13955 14127 14211 14628 14629 14768 15084 15153 15317 16369 16610 16775 16806 16852 17202 230: 10505 10556 10635 10672 10759 10860 10946 10986 10998 11200 11241 11316 11328 11351 11407 11476 11477 11482 11483 11555 11558 11560 11575 11577 11580 11581 11628 11782 11795 11821 11849 11865 11949 12072 12099 12102 12106 12108 12126 12127 12129 12132 12133 12134 12137 12142 12149 12178 12193 12320 12324 12344 12391 12422 12443 12483 12484 12528 12534 12540 12551 12599 12619 12692 12839 12878 13138 13151 13245 13246 13273 13288 13307 13311 13320 13474 13571 13626 13632 13634 13639 13681 13686 13689 13769 13831 13848 13871 13911 13929 14026 14044 14087 14412 14441 14442 14493 14517 14518 14588 14641 14658 14706 14708 14731 14828 14834 14882 14889 14936 15062 15064 15228 15281 15283 15284 15289 15291 15312 15316 15319 15322 15445 15553 15598 15673 15695 15778 15830 15908 15913 15940 16046 16052 16074 16080 16085 16183 16218 16296 16316 16337 16423 16722 17135 17163 17343 17356 17375 231: 10703 10903 11022 11090 11320 11551 11930 11982 11993 12004 12298 12417 12758 12851 12952 13270 13332 13387 14025 14050 14089 14305 14627 14941 14960 15075 15184 15454 15517 15627 15643 15671 15733 15760 15767 15768 15829 15916 15994 16179 16308 16468 16621 16708 16927 17211 17316 232: 10543 10800 11044 14341 14390 14733 15514 15611 16225 17148 233: 10592 10722 10908 10940 10944 11055 11125 11374 11481 11539 11892 12026 12448 12491 12598 12680 12799 12834 12838 13098 13304 13531 13691 13886 14023 14625 14760 14802 14838 14933 15103 15164 15192 15195 15853 16041 16077 16091 16107 16728 16762 17369 234: 10763 10970 11270 11299 11382 11620 11648 11868 12202 12691 12701 12841 12853 12986 13078 13430 13611 13832 13961 14045 14198 14577 14900 14997 15300 15402 15546 15874 15903 15910 15920 16035 16151 16410 16596 16638 16689 16769 235: 10495 10497 10499 10534 10546 10558 10576 10577 10587 10646 10651 10671 10675 10742 10743 10746 10785 10788 10807 10817 10826 10833 10844 10849 10859 10861 10870 10874 10884 10887 10895 10898 10902 10913 10923 10928 10935 10941 10951 10972 11007 11028 11067 11071 11078 11103 11104 11112 11115 11129 11139 11174 11180 11189 11191 11192 11196 11209 11213 11224 11230 11247 11256 11257 11262 11302 11307 11313 11319 11334 11370 11373 11437 11447 11473 11474 11489 11513 11527 11550 11556 11576 11600 11619 11623 11627 11632 11686 11696 11702 11704 11716 11721 11728 11734 11751 11752 11761 11790 11793 11805 11811 11813 11820 11824 11835 11845 11848 11872 11882 11921 11927 11928 11934 11942 11947 11948 11961 11962 11963 11974 11978 11979 12000 12003 12021 12045 12069 12117 12119 12170 12194 12197 12210 12214 12232 12246 12250 12254 12262 12273 12274 12293 12307 12318 12330 12350 12354 12356 12365 12372 12389 12396 12430 12446 12471 12494 12495 12503 12505 12519 12525 12556 12567 12571 12573 12574 12587 12597 12614 12617 12623 12639 12665 12687 12700 12721 12734 12749 12752 12755 12785 12798 12807 12814 12847 12849 12852 12863 12905 12913 12959 12962 12966 12973 12994 13006 13025 13026 13060 13081 13095 13129 13130 13167 13184 13200 13225 13231 13256 13267 13269 13293 13309 13310 13319 13326 13341 13352 13415 13417 13427 13433 13469 13487 13525 13527 13530 13532 13535 13569 13574 13587 13589 13594 13615 13620 13623 13655 13705 13714 13740 13741 13742 13764 13765 13768 13774 13775 13776 13779 13788 13792 13811 13815 13828 13830 13852 13860 13873 13875 13877 13881 13885 13948 13982 13990 13995 14004 14006 14009 14028 14029 14033 14048 14049 14053 14072 14084 14086 14095 14116 14122 14130 14144 14160 14164 14177 14196 14252 14257 14267 14269 14279 14281 14304 14350 14360 14385 14433 14445 14446 14451 14477 14494 14501 14511 14513 14525 14556 14585 14595 14605 14620 14666 14669 14673 14702 14767 14787 14798 14804 14818 14833 14864 14895 14930 14940 14951 14954 14972 14973 14977 14985 14994 15004 15010 15011 15073 15074 15085 15086 15099 15106 15127 15131 15151 15152 15170 15187 15218 15249 15259 15270 15273 15277 15320 15329 15358 15369 15470 15477 15486 15487 15506 15507 15566 15610 15640 15650 15660 15675 15688 15691 15697 15707 15714 15719 15747 15753 15755 15756 15757 15758 15759 15770 15837 15857 15885 15905 15925 15960 15968 15982 15997 15998 16008 16045 16065 16089 16114 16117 16136 16155 16210 16228 16239 16244 16249 16274 16276 16304 16336 16351 16376 16383 16391 16399 16406 16407 16413 16416 16418 16440 16449 16478 16489 16506 16513 16522 16531 16555 16581 16634 16664 16703 16704 16743 16817 16846 16887 16904 16918 16922 16925 16928 16932 16954 16955 16967 16970 16973 16984 16993 16997 17004 17020 17024 17025 17032 17044 17048 17099 17117 17118 17122 17127 17147 17158 17164 17173 17200 17217 17235 17263 17274 17301 17315 17319 17325 17341 17344 236: 11742 12406 237: 11223 11832 12406 12559 12911 16502 238: 10840 11211 11713 11815 11817 11819 11893 12181 12185 12192 12388 12776 13286 13453 13667 13772 14047 14090 14150 14532 14562 14566 14603 14647 14664 14743 14763 14779 14860 14867 14934 15007 15032 15364 15586 15831 16134 16195 16270 16286 16301 16706 16707 16749 16784 16886 16986 17033 17035 17039 17060 17121 17264 17293 17350 17362 239: 10563 10611 10639 10643 10647 10677 10793 10955 11040 11046 11147 11238 11718 11898 12150 12281 12329 12341 12641 12651 12774 12883 12926 12999 13097 13099 13371 13604 13835 13964 14106 14112 14131 14372 14547 14597 14613 14756 14970 15194 15232 15341 15345 15628 15669 16064 16198 16203 16205 16271 16691 16837 17012 17055 17132 17284 240: 10529 10602 10606 10629 10707 10728 10752 10760 10779 10825 10831 10853 10872 10893 11027 11108 11159 11245 11315 11377 11420 11427 11454 11499 11559 11583 11689 11701 11794 11853 11857 11946 11973 12020 12081 12201 12253 12570 12882 12893 12910 12945 12948 12975 12976 12978 12979 12982 12996 12997 13000 13001 13002 13004 13008 13038 13040 13041 13042 13302 13323 13439 13603 13680 13736 13739 13744 13746 13796 13840 13939 13984 14100 14168 14236 14326 14334 14401 14522 14551 14582 14601 14602 14608 14632 14634 14688 14797 14809 14845 14886 14920 15112 15130 15292 15293 15339 15344 15351 15382 15383 15384 15385 15387 15401 15509 15519 15522 15538 15545 15549 15551 15552 15555 15580 15587 15589 15591 15609 15634 15636 15663 15666 15687 15690 15692 15718 15722 15745 15763 15765 15787 15796 15799 15804 15842 15867 15893 15926 15929 16042 16079 16130 16133 16135 16142 16159 16172 16221 16278 16326 16331 16334 16371 16374 16420 16439 16473 16526 16548 16549 16550 16551 16584 16615 16747 16934 17013 17059 17079 17080 17083 241: 10582 10601 10813 10945 11279 11547 11675 11736 12355 12482 12590 12609 12909 12939 13034 13102 13105 13121 13306 13679 13718 13842 13922 13962 14000 14212 14319 14367 14526 14564 14590 14749 14854 14871 14938 14998 15098 15111 15413 15523 15661 15771 15775 15935 16213 16580 16609 17090 17116 17139 17237 17275 17288 17310 17355 242: 10481 10510 10522 10548 10555 10560 10627 10640 10649 10653 10680 10686 10692 10721 10724 10727 10731 10733 10735 10737 10739 10757 10758 10812 10899 10958 10962 10967 10969 10971 10987 10989 10993 10995 10997 11023 11050 11111 11244 11255 11266 11306 11308 11309 11312 11314 11336 11337 11338 11339 11340 11341 11342 11345 11361 11363 11364 11365 11366 11367 11369 11385 11389 11390 11392 11395 11397 11398 11399 11401 11416 11417 11423 11426 11430 11435 11439 11455 11457 11461 11464 11466 11491 11492 11493 11495 11496 11512 11515 11518 11522 11524 11526 11530 11563 11603 11625 11656 11685 11750 11766 11913 11985 11986 12006 12019 12022 12036 12039 12040 12044 12049 12052 12074 12077 12080 12082 12084 12096 12097 12098 12101 12103 12104 12107 12110 12114 12118 12136 12140 12141 12147 12148 12152 12154 12157 12158 12159 12160 12177 12180 12184 12187 12189 12198 12209 12212 12215 12218 12219 12221 12224 12238 12240 12242 12244 12247 12249 12251 12252 12261 12264 12265 12267 12269 12272 12285 12288 12291 12297 12299 12301 12304 12312 12319 12345 12346 12348 12368 12370 12373 12376 12380 12429 12470 12474 12501 12502 12504 12509 12605 12613 12628 12669 12670 12672 12675 12696 12697 12698 12710 12711 12725 12726 12728 12730 12731 12739 12753 12756 12763 12781 12784 12795 12797 12801 12802 12808 12810 12818 12837 12840 12842 12846 12848 12861 12862 12864 12865 12867 12892 12894 12895 12898 12899 12902 12920 12924 12925 12927 12928 12944 12949 12951 12953 12956 12958 12984 12985 13024 13028 13032 13052 13055 13075 13080 13082 13091 13096 13109 13112 13116 13117 13120 13123 13124 13125 13127 13128 13143 13146 13148 13150 13156 13159 13162 13163 13164 13175 13176 13179 13186 13189 13190 13193 13196 13198 13199 13202 13212 13219 13222 13223 13229 13230 13232 13234 13235 13237 13249 13346 13403 13425 13452 13455 13458 13481 13488 13491 13500 13506 13519 13521 13524 13534 13540 13548 13557 13558 13560 13563 13570 13576 13577 13579 13581 13583 13586 13591 13597 13601 13606 13614 13616 13619 13622 13630 13641 13642 13647 13654 13660 13663 13666 13670 13676 13677 13700 13704 13707 13711 13712 13722 13723 13724 13726 13728 13729 13735 13738 13748 13752 13756 13760 13762 13780 13782 13787 13789 13791 13810 13813 13816 13818 13821 13839 13844 13847 13849 13850 13869 13895 13916 13941 13944 13947 13950 13951 13969 13972 13974 13978 13979 13980 13996 13998 14002 14003 14008 14012 14030 14032 14035 14036 14039 14043 14054 14058 14061 14063 14065 14078 14163 14251 14266 14268 14274 14293 14295 14328 14331 14333 14336 14337 14339 14342 14365 14370 14374 14376 14377 14379 14384 14387 14391 14408 14411 14414 14418 14420 14421 14423 14427 14443 14447 14448 14450 14452 14457 14459 14460 14462 14463 14469 14476 14483 14489 14491 14660 14711 14717 14741 14752 14765 14770 14771 14781 14827 14869 14981 14983 14986 14990 14991 15058 15239 15264 15267 15269 15276 15280 15282 15286 15298 15299 15304 15305 15311 15313 15350 15357 15361 15366 15371 15386 15390 15393 15396 15400 15410 15419 15422 15427 15429 15433 15451 15452 15458 15459 15462 15479 15493 15495 15498 15501 15503 15508 15525 15527 15530 15533 15534 15541 15542 15543 15544 15557 15560 15562 15568 15572 15612 15616 15619 15632 15639 15642 15657 15672 15685 15696 15700 15705 15727 15728 15779 15784 15789 15791 15794 15802 15810 15813 15865 15868 15892 15896 15899 15923 15953 15955 15957 15959 15984 15986 15987 15988 15990 15991 15992 15993 15995 15996 16021 16023 16026 16027 16028 16030 16031 16032 16033 16034 16048 16051 16053 16054 16055 16056 16058 16061 16062 16063 16067 16069 16090 16093 16094 16096 16097 16098 16099 16100 16102 16103 16104 16105 16106 16111 16121 16123 16125 16127 16128 16129 16182 16241 16242 16243 16247 16248 16252 16253 16254 16258 16267 16269 16277 16279 16282 16283 16285 16288 16293 16303 16305 16306 16310 16313 16319 16322 16324 16325 16329 16330 16345 16350 16356 16359 16361 16362 16363 16373 16379 16381 16388 16389 16394 16415 16417 16422 16424 16427 16428 16431 16433 16438 16443 16453 16455 16457 16460 16464 16466 16488 16490 16495 16498 16509 16512 16514 16518 16528 16530 16533 16536 16537 16539 16542 16543 16546 16561 16570 16571 16579 16583 16588 16727 16733 16778 16779 16780 16781 16783 16792 16793 16795 16797 16798 16801 16803 16804 16805 16808 16811 16812 16813 16826 16829 16831 16833 16835 16836 16838 16840 16841 16843 16844 16867 16868 16870 16872 16873 16876 16878 16882 16883 16884 16885 16905 16907 16913 16931 16933 16935 16936 16937 16943 16964 16966 16968 16969 16971 17029 17037 17054 17056 17057 17058 17064 17065 17071 17073 17076 17082 17085 17087 17088 17089 17094 17098 17103 17106 17109 17111 17124 17126 17130 17133 17134 17138 17140 17142 17146 17152 17165 17167 17170 17172 17174 17177 17181 17183 17186 17187 17190 17193 17196 17206 17209 17210 17213 17216 17218 17223 17224 17227 17228 17255 17256 17258 17259 17266 17272 17287 17290 17291 17297 17320 17324 17326 17330 17333 17335 17336 17337 17340 17367

Recombinant DNA constructs are prepared using the DNA encoding each of the identified homologs and the constructs are used to prepare multiple events of transgenic corn, soybean, canola and cotton plants as illustrated in Examples 2-5. Plants are regenerated from the transformed plant cells and used to produce progeny plants and seed that are screened for enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil. From each group of multiple events of transgenic plants with a specific recombinant DNA for a homolog the event that produces the greatest enhancement in yield, water use efficiency, nitrogen use efficiency, enhanced cold tolerance, enhanced seed protein and enhanced seed oil is identified and progeny seed is selected for commercial development.

Example 7 Consensus Sequence

This example illustrates the identification of consensus amino acid sequence for the proteins and homologs encoded by DNA that is used to prepare the transgenic seed and plants of this invention having enhanced agronomic traits.

ClustalW program was selected for multiple sequence alignments of the amino acid sequence of SEQ ID NO: 122, 133, 136, 138, 141, 143, 153, 155, 173-174, 182, 197, 203, 206, 208, 215, 217, 229, 234, 236-237, 240 and their homologs. Three major factors affecting the sequence alignments dramatically are (1) protein weight matrices; (2) gap open penalty; (3) gap extension penalty. Protein weight matrices available for ClustalW program include Blosum, Pam and Gonnet series. Those parameters with gap open penalty and gap extension penalty were extensively tested. On the basis of the test results, Blosum weight matrix, gap open penalty of 10 and gap extension penalty of 1 were chosen for multiple sequence alignment.

The consensus amino acid sequence can be used to identify DNA corresponding to the full scope of this invention that is useful in providing transgenic plants, for example corn and soybean plants with enhanced agronomic traits, for example improved nitrogen use efficiency, improved yield, improved water use efficiency and/or improved growth under cold stress, due to the expression in the plants of DNA suppressing a protein with amino acid sequence identical to the consensus amino acid sequence.

The SEQ ID NOs for the identified consensus sequences are reported in table 8 below and the full consensus sequences are provided in the attached sequence listing.

TABLE 8 Consensus Gene ID PEP SEQ ID NO SEQ ID NO Mnom000132-Mnom000134 122 17381 Mnom000451 133 17382 Mnom000481 136 17383 Mnom000486 138 17384 Mnom000494 141 17385 Mnom000497 143 17386 Mnom000636 153 17387 Mnom000640 155 17388 Mnom000766-Mnom000767 173 17389 Mnom000822-Mnom000823 174 17390 Mnom000986 182 17391 PHE0002862 197 17392 PHE0004853 203 17393 PHE0006378 206 17394 PHE0006496 208 17395 PHE0008407 215 17396 PHE0008541 217 17397 PHE0011028 229 17398 PHE0013205 234 17399 PHE0013712 236 17400 PHE0013713 237 17401 TRG0000291 240 17402

Example 9 Identification of Amino Acid Domain by Pfam Analysis

This example illustrates the identification of domain and domain module by Pfam analysis.

The amino acid sequence of the expressed proteins that are shown to be associated with an enhanced trait were analyzed for Pfam protein family against the current Pfam collection of multiple sequence alignments and hidden Markov models using the HMMER software in the appended computer listing. The Pfam protein domains and modules for the proteins of SEQ ID NOs: 123-132, 134-135, 137, 139-140, 142, 145-152, 154, 156-172, 175-181, 183-196, 198-202, 204-205, 207, 209-211, 213, 216, 218-228, 230-233, 235, 238-239, and 241-242 are shown in Tables 9, 10 and 11. The Hidden Markov model databases for the identified patent families are also in the appended computer listing allowing identification of other homologous proteins and their cognate encoding DNA to enable the full breadth of the invention for a person of ordinary skill in the art. Certain proteins are identified by a single Pfam domain and others by multiple Pfam domains.

TABLE 9 Pfam annotation PEP SEQ ID NO Gene ID Pfam domain name Begin Stop Score E-value 123 Mnom000202 Copine 116 264 331.7 1.50E−96 124 Mnom000215-Mnom000216 CBFB_NFYA 97 153 143.1 8.50E−40 125 Mnom000219-Mnom000220 CBFD_NFYB_HMF 25 90 124.7 3.10E−34 126 Mnom000256-Mnom000257 Glu_synthase 115 494 683.3 2.00E−202 127 Mnom000260-Mnom000261 HEAT 183 219 22.6 0.0016 127 Mnom000260-Mnom000261 HEAT 716 753 17.5 0.057 127 Mnom000260-Mnom000261 FAT 1435 1801 483.5 2.90E−142 127 Mnom000260-Mnom000261 Rapamycin_bind 1908 2007 232.8 8.80E−67 127 Mnom000260-Mnom000261 PI3_PI4_kinase 2074 2324 370.7 2.70E−108 127 Mnom000260-Mnom000261 FATC 2432 2464 65.9 1.60E−16 128 Mnom000262 F-box 29 76 36.2 1.30E−07 129 Mnom000324 AP2 136 188 81.9 2.30E−21 130 Mnom000372 SAP 34 68 45.6 1.90E−10 130 Mnom000372 zf-MIZ 357 406 111.3 3.20E−30 131 Mnom000412-Mnom000413 14-3-3 4 219 277.4 3.20E−80 132 Mnom000444 GATase_2 39 409 772.4 3.20E−229 132 Mnom000444 Glu_syn_central 483 784 680.2 1.80E−201 132 Mnom000444 Glu_synthase 841 1226 867.4 8.10E−258 132 Mnom000444 GXGXG 1304 1499 387.2 2.90E−113 134 Mnom000471 Glyco_transf_20 16 482 1010.3 7.40E−301 134 Mnom000471 Trehalose_PPase 572 819 382.5 7.50E−112 135 Mnom000480 Response_reg 16 128 26.4 5.90E−06 137 Mnom000485 FAR1 67 160 131.4 2.90E−36 137 Mnom000485 FART 204 298 126.8 6.90E−35 139 Mnom000490 PTR2 1 257 46.1 1.30E−10 140 Mnom000491 PPR 151 185 14.4 0.18 140 Mnom000491 PPR 186 219 11.9 0.35 140 Mnom000491 PPR 220 254 5.4 2 140 Mnom000491 PPR 258 292 18.4 0.029 140 Mnom000491 PPR 293 327 25.4 0.00024 140 Mnom000491 PPR 328 362 26.1 0.00014 140 Mnom000491 PPR 363 397 32 2.40E−06 140 Mnom000491 PPR 398 432 12.1 0.33 140 Mnom000491 PPR 434 468 23.7 0.00076 140 Mnom000491 PPR 469 503 29.4 1.50E−05 140 Mnom000491 PPR 534 568 34.5 4.20E−07 142 Mnom000496 efhand 109 137 23.6 0.00082 145 Mnom000517 14-3-3 8 225 391.1 1.90E−114 146 Mnom000554 DUF761 9 329 302.9 6.80E−88 147 Mnom000570 SAP 11 45 49.3 1.50E−11 147 Mnom000570 PHD 114 168 35.4 2.20E−07 147 Mnom000570 zf-MIZ 359 408 88.1 3.10E−23 148 Mnom000580 HSP20 133 230 144.1 4.20E−40 149 Mnom000616-Mnom000617 Fer2 19 88 47.5 5.30E−11 149 Mnom000616-Mnom000617 Fer2_2 98 173 127.1 5.70E−35 149 Mnom000616-Mnom000617 FAD_binding_5 260 440 273.4 5.10E−79 149 Mnom000616-Mnom000617 CO_deh_flav_C 447 558 151.7 2.20E−42 149 Mnom000616-Mnom000617 Ald_Xan_dh_C 612 719 168.1 2.60E−47 149 Mnom000616-Mnom000617 Ald_Xan_dh_C2 727 1272 756.1 2.50E−224 150 Mnom000618-Mnom000619 Fer2 11 80 42.5 1.70E−09 150 Mnom000618-Mnom000619 Fer2_2 90 165 126.5 8.50E−35 150 Mnom000618-Mnom000619 FAD_binding_5 252 432 271.6 1.80E−78 150 Mnom000618-Mnom000619 CO_deh_flav_C 439 550 145.5 1.60E−40 150 Mnom000618-Mnom000619 Ald_Xan_dh_C 604 711 159.3 1.20E−44 150 Mnom000618-Mnom000619 Ald_Xan_dh_C2 719 1264 746.6 1.90E−221 151 Mnom000626-Mnom000627 2OG-FeII_Oxy 180 280 121.3 3.10E−33 152 Mnom000628 2OG-Fell_Oxy 180 280 121.3 3.10E−33 154 Mnom000637 B3 312 417 118.5 2.30E−32 154 Mnom000637 Auxin_resp 439 524 160.7 4.30E−45 154 Mnom000637 AUX_IAA 681 826 −74.9 0.00048 156 Mnom000641 XRN_N 1 256 589.7 3.10E−174 156 Mnom000641 zf-CCHC 263 280 28.9 8.30E−06 157 Mnom000643 PALP 36 329 341 2.30E−99 158 Mnom000645 DAO 164 518 −26.1 0.00058 158 Mnom000645 GMC_oxred_N 210 434 358.8 1.00E−104 159 Mnom000647 Peptidase_S10 42 455 562.2 6.00E−166 160 Mnom000682 Glycos_transf_1 296 459 121.8 2.20E−33 161 Mnom000690 Redoxin 76 232 56.6 9.30E−14 161 Mnom000690 AhpC-TSA 77 211 214 4.10E−61 161 Mnom000690 1-cysPrx_C 221 265 64.5 4.10E−16 162 Mnom000107 PAS_2 70 186 231.5 2.10E−66 162 Mnom000107 GAF 219 404 105.2 2.10E−28 162 Mnom000107 Phytochrome 415 595 408.4 1.20E−119 162 Mnom000107 PAS 622 737 93.7 6.60E−25 162 Mnom000107 PAS_4 628 742 18.7 0.0033 162 Mnom000107 PAS 752 877 101.1 3.70E−27 162 Mnom000107 HisKA 897 961 27.7 4.90E−05 162 Mnom000107 HATPase_c 1011 1123 67.2 6.00E−17 163 Mnom000108 PAS_2 70 186 231.5 2.10E−66 163 Mnom000108 GAF 219 404 108.6 2.10E−29 163 Mnom000108 Phytochrome 415 595 408.4 1.20E−119 163 Mnom000108 PAS 622 737 93.7 6.60E−25 163 Mnom000108 PAS_4 628 742 18.7 0.0033 163 Mnom000108 PAS 752 877 101.1 3.70E−27 163 Mnom000108 HisKA 897 961 27.7 4.90E−05 163 Mnom000108 HATPase_c 1011 1123 67.2 6.00E−17 164 Mnom000109 PAS_2 70 186 231.5 2.10E−66 164 Mnom000109 GAF 219 404 101.3 3.20E−27 164 Mnom000109 Phytochrome 415 595 408.4 1.20E−119 164 Mnom000109 PAS 622 737 93.7 6.60E−25 164 Mnom000109 PAS_4 628 742 18.7 0.0033 164 Mnom000109 PAS 752 877 101.1 3.70E−27 164 Mnom000109 HisKA 897 961 27.7 4.90E−05 164 Mnom000109 HATPase_c 1011 1123 67.2 6.00E−17 165 Mnom000110 PAS_2 97 217 213.1 7.30E−61 165 Mnom000110 GAF 250 432 111.1 3.80E−30 165 Mnom000110 Phytochrome 443 622 414.3 1.90E−121 165 Mnom000110 PAS 653 769 123.6 6.20E−34 165 Mnom000110 PAS_4 659 774 28.3 3.20E−05 165 Mnom000110 PAS 784 906 89.3 1.30E−23 165 Mnom000110 HisKA 926 990 53.4 8.90E−13 165 Mnom000110 HATPase_c 1038 1150 50.3 7.40E−12 166 Mnom000111 PAS_2 97 217 213.1 7.30E−61 166 Mnom000111 GAF 250 432 114.7 3.00E−31 166 Mnom000111 Phytochrome 443 622 414.3 1.90E−121 166 Mnom000111 PAS 1653 769 123.6 6.20E−34 166 Mnom000111 PAS_4 659 774 28.3 3.20E−05 166 Mnom000111 PAS 784 906 89.3 1.30E−23 166 Mnom000111 HisKA 926 990 53.4 8.90E−13 166 Mnom000111 HATPase_c 1038 1150 50.3 7.40E−12 167 Mnom000112 PAS_2 97 217 213.1 7.30E−61 167 Mnom000112 GAF 250 432 110.1 7.50E−30 167 Mnom000112 Phytochrome 443 622 414.3 1.90E−12 167 Mnom000112 PAS 653 769 123.6 6.20E−34 167 Mnom000112 PAS_4 659 774 28.3 3.20E−05 167 Mnom000112 PAS 784 906 89.3 1.30E−23 167 Mnom000112 HisKA 926 990 53.4 8.90E−13 167 Mnom000112 HATPase_c 1038 1150 50.3 7.40E−12 168 Mnom000113, PAS_2 70 186 231.5 2.10E−66 Mnom000116 168 Mnom000113, GAF 219 404 102.6 1.40E−27 Mnom000116 168 Mnom000113, Phytochrome 415 595 408.4 1.20E−119 Mnom000116 168 Mnom000113, PAS 622 737 93.7 6.60E−25 Mnom000116 168 Mnom000113, PAS_4 628 742 18.7 0.0033 Mnom000116 168 Mnom000113, PAS 752 877 101.1 3.70E−27 Mnom000116 168 Mnom000113, HisKA 897 961 27.7 4.90E−05 Mnom000116 168 Mnom000113, HATPase_c 1011 1123 67.2 6.00E−17 Mnom000116 169 Mnom000114, PAS_2 97 217 213.1 7.30E−61 Mnom000117 169 Mnom000114, GAF 250 432 112.6 1.30E−30 Mnom000117 169 Mnom000114, Phytochrome 443 622 414.3 1.90E−121 Mnom000117 169 Mnom000114, PAS 653 769 123.6 6.20E−34 Mnom000117 169 Mnom000114, PAS_4 659 774 28.3 3.20E−05 Mnom000117 169 Mnom000114, PAS 784 906 89.3 1.30E−23 Mnom000117 169 Mnom000114, HisKA 926 990 53.4 8.90E−13 Mnom000117 169 Mnom000114, HATPase_c 1038 1150 50.3 7.40E−12 Mnom000117 170 Mnom000115, PAS_2 97 217 213.1 7.30E−61 Mnom000118 170 Mnom000115, GAF 250 432 115.9 1.30E−31 Mnom000118 170 Mnom000115, Phytochrome 443 622 414.3 1.90E−121 Mnom000118 170 Mnom000115, PAS 653 769 123.6 6.20E−34 Mnom000118 170 Mnom000115, PAS_4 659 774 28.3 3.20E−05 Mnom000118 170 Mnom000115, PAS 784 906 89.3 1.30E−23 Mnom000118 170 Mnom000115, HisKA 926 990 53.4 8.90E−13 Mnom000118 170 Mnom000115, HATPase_c 1038 1150 50.3 7.40E−12 Mnom000118 171 Mnom000119 PAS_2 70 186 231.5 2.10E−66 171 Mnom000119 GAF 219 404 107 6.20E−29 171 Mnom000119 Phytochrome 415 595 408.4 1.20E−119 171 Mnom000119 PAS 622 737 93.7 6.60E−25 171 Mnom000119 PAS_4 628 742 18.7 0.0033 171 Mnom000119 PAS 752 877 101.1 3.70E−27 171 Mnom000119 HisKA 897 961 27.7 4.90E−05 171 Mnom000119 HATPase_c 1011 1123 67.2 6.00E−17 172 Mnom000738 DUF260 7 108 203.5 5.70E−58 175 Mnom000857, DUF231 364 542 272.2 1.20E−78 Mnom000858, Mnom000860 176 Mnom000873 Glyco_hydro_18 27 306 45.5 1.70E−10 177 Mnom000886 Synaptobrevin 132 238 56.1 1.40E−13 178 Mnom000923-Mnom000924 PAS_2 97 217 213.1 7.30E−61 178 Mnom000923-Mnom000924 GAF 250 432 115.9 1.30E−31 178 Mnom000923-Mnom000924 Phytochrome 443 622 406.8 3.60E−119 178 Mnom000923-Mnom000924 PAS 653 769 123.6 6.20E−34 178 Mnom000923-Mnom000924 PAS_4 659 774 28.3 3.20E−05 178 Mnom000923-Mnom000924 PAS 784 906 89.3 1.30E−23 178 Mnom000923-Mnom000924 HisKA 926 990 53.4 8.90E−13 178 Mnom000923-Mnom000924 HATPase_c 1038 1150 50.3 7.40E−12 179 Mnom000937 PAS 116 230 22.8 0.0014 179 Mnom000937 PAS_3 141 227 19.9 0.0011 179 Mnom000937 PAS 390 504 10.5 0.047 179 Mnom000937 Pkinase 582 870 291.4 2.00E−84 179 Mnom000937 Pkinase_Tyr 582 877 −58.7 8.00E−10 180 Mnom000942, TP6A_N 89 156 84.6 3.50E−22 Mnom000955-Mnom000956 181 Mnom000985 Response_reg 16 128 26.4 5.90E−06 183 Mnom001055 Peptidase_S10 42 455 562.2 6.00E−166 184 Mnom001090-Mnom001092 Gln-synt_N 31 111 149.1 1.30E−41 184 Mnom001090-Mnom001092 Gln-synt_C 117 369 295.8 9.50E−86 185 Mnom001138 UDPGP 26 443 913.5 1.00E−271 186 Mnom001140 DnaJ 11 80 75 2.80E−19 187 Mnom001141 DnaJ 12 78 109 1.60E−29 188 Mnom001142 NAPRTase 175 439 150.5 5.10E−42 189 Mnom001146 DUF296 114 234 180.6 4.40E−51 190 Mnom001147 DUF296 114 234 180.6 4.40E−51 191 Mnom001160 zf-A20 14 38 34.2 5.10E−07 191 Mnom001160 zf-AN1 115 155 72.8 1.20E−18 192 Mnom001162 Cellulose_synt 284 1107 1889.4 0 193 PHE0000597 Mannitol_dh 10 132 181.9 1.80E−51 193 PHE0000597 Mannitol_dh_C 157 383 394.4 1.90E−115 194 PHE0001556 RimK 226 486 −20.7 1.20E−05 194 PHE0001556 Mur_ligase_M 498 711 138.1 2.70E−38 194 PHE0001556 Mur_ligase_C 735 821 75.8 1.60E−19 195 PHE0001557 RimK 217 370 −48.2 0.0014 195 PHE0001557 Mur_ligase_M 491 704 138.5 2.10E−38 195 PHE0001557 Mur_ligase_C 728 813 80.8 5.00E−21 196 PHE0001559 Peptidase_S51 52 212 44.4 4.40E−10 198 PHE0003348 HLH 310 359 70 8.90E−18 199 PHE0003706 SSF 54 471 −94.5 2.00E−05 200 PHE0003708 SSF 46 463 −112.6 8.80E−05 201 PHE0004230 E2F_TDP 227 292 134 4.90E−37 202 PHE0004820 HLH 113 162 62.1 2.10E−15 204 PHE0006017 Na_H_Exchanger 26 445 173.4 6.40E−49 205 PHE0006102 Brix 27 342 311.1 2.30E−90 207 PHE0006380 RNase_PH_C 129 201 61.9 2.40E−15 209 PHE0006610 GSHPx 119 227 234.2 3.30E−67 210 PHE0006613 GSHPx 29 137 200.6 4.10E−57 211 PHE0006916 RAMP4 49 112 130 7.80E−36 213 PHE0007643 LRRNT_2 38 79 39.3 1.60E−08 213 PHE0007643 LRR_1 155 177 12.5 1.2 213 PHE0007643 LRR_1 179 201 15 0.31 213 PHE0007643 LRR_1 227 249 16.4 0.12 213 PHE0007643 Pkinase_Tyr 429 696 0.6 9.10E−14 216 PHE0008411 DUF778 41 162 269.1 1.00E−77 218 PHE0008602 Gln-synt_N 103 184 168.6 1.80E−47 218 PHE0008602 Gln-synt_C 191 472 579.1 5.00E−171 219 PHE0009339 HSP70 66 673 1343.3 0 220 PHE0009341 TrkH 211 569 125.4 1.80E−34 221 PHE0010195 PTR2 85 432 70 8.70E−18 222 PHE0010352 CBFD_NFYB_HMF 31 96 132.4 1.40E−36 223 PHE0010354 CBFD_NFYB_HMF 34 99 128.8 1.80E−35 224 PHE0010646 Spermine_synth 46 291 500.6 2.10E−147 225 PHE0010648 SNF2_N 25 373 −19.4 1.30E−07 225 PHE0010648 Helicase_C 441 520 59.4 1.40E−14 226 PHE0010649 ResIII 18 230 2.1 3.60E−05 226 PHE0010649 SNF2_N 24 374 4.4 5.60E−09 226 PHE0010649 Helicase_C 442 521 56.7 8.90E−14 227 PHE0010852 HLH 403 451 40.3 7.80E−09 228 PHE0010956 Nucleoside_tran 167 450 514.2 1.70E−151 230 PHE0011063 Globin 49 145 53 1.20E−12 231 PHE0011543 zf-B_box 4 50 27.5 5.50E−05 232 PHE0013202 HLH 172 221 22.2 0.00075 233 PHE0013203 SAM_2 236 302 54.6 3.70E−13 233 PHE0013203 SAM_1 237 300 85.7 1.60E−22 233 PHE0013203 DRMBL 609 707 160.2 6.20E−45 235 PHE0013292 PDZ 220 304 34.6 4.00E−07 235 PHE0013292 Peptidase_S41 340 503 244.8 2.20E−70 238 PHE0013734 SRF-TF 9 59 64.9 3.00E−16 239 TRG0000049 CBFD_NFYB_HMF 75 140 100.2 7.10E−27 241 TRG0000332 CBFB_NFYA 192 248 141.2 3.30E−39 242 1071693 PAS_2 98 218 192.3 1.30E−54 242 1071693 GAF 251 433 116.9 6.70E−32 242 1071693 Phytochrome 444 623 409.1 7.40E−120 242 1071693 PAS 654 738 30.7 5.80E−06

TABLE 10 Pfam module annotation PEP SEQ ID NO Gene ID Pfam domain module Position 123 Mnom000202 Copine 116-264 124 Mnom000215-Mnom000216 CBFB_NFYA  97-153 125 Mnom000219-Mnom000220 CBFD_NFYB_HMF 25-90 126 Mnom000256-Mnom000257 Glu_synthase 115-494 127 Mnom000260-Mnom000261 HEAT::HEAT::FAT::Rapamycin_bind:: 183-219::716-753::1435-1801:: PI3_PI4_kinase::FAT C 1908-2007::2074-2324:: 2432-2464 128 Mnom000262 F-box 29-76 129 Mnom000324 AP2 136-188 130 Mnom000372 SAP::zf-MIZ 34-68::357-406 131 Mnom000412-Mnom000413 14-3-3  4-219 132 Mnom000444 GATase_2::Glu_syn_central::Glu_synthase:: 39-409::483-784::841-1226:: GXGXG 1304-1499 134 Mnom000471 Glyco_transf_20::Trehalose_PPase 16-482::572-819 135 Mnom000480 Response_reg  16-128 137 Mnom000485 FAR1::FAR1 67-160::204-298 139 Mnom000490 PTR2  1-257 140 Mnom000491 PPR::PPR::PPR::PPR::PPR::PPR:: 151-185::186-219::220-254:: PPR::PPR::PPR::PPR::PPR 258-292::293-327::328-362:: 363-397::398-432::434-468:: 469-503::534-568 142 Mnom000496 Efhand 109-137 145 Mnom000517 14-3-3  8-225 146 Mnom000554 DUF761  9-329 147 Mnom000570 SAP::PHD::zf-MIZ 11-45::114-168::359-408 148 Mnom000580 HSP20 133-230 149 Mnom000616-Mnom000617 Fer2::Fer2_2::FAD_binding_5:: 19-88::98-173::260-440::447-558:: CO_deh_flav_C::Ald_Xan_dh_C:: 612-719::727-1272 Ald_Xan_dh_C2 150 Mnom000618-Mnom000619 Fer2::Fer2_2::FAD_binding_5:: 11-80::90-165::252-432::439-550:: CO_deh_flav_C::Ald_Xan_dh_C:: 604-711::719-1264 Ald_Xan_dh_C2 151 Mnom000626-Mnom000627 2OG-FeII_Oxy 180-280 152 Mnom000628 2OG-FeII_Oxy 180-280 154 Mnom000637 B3::Auxin_resp::AUX_IAA 312-417::439-524::681-826 156 Mnom000641 XRN_N::zf-CCHC 1-256::263-280 157 Mnom000643 PALP  36-329 158 Mnom000645 GMC_oxred_N 210-434 159 Mnom000647 Peptidase_S10  42-455 160 Mnom000682 Glycos_transf_1 296-459 161 Mnom000690 AhpC-TSA::1-cysPrx_C 77-211::221-265 162 Mnom000107 PAS_2::GAF::Phytochrome::PAS:: 70-186::219-404::415-595:: PAS::HisKA::HATPase_c 622-737::752-877::897-961::1011-1123 163 Mnom000108 PAS_2::GAF::Phytochrome::PAS:: 70-186::219-404::415-595:: PAS::HisKA::HATPase_c 622-737::752-877::897-961::1011-1123 164 Mnom000109 PAS_2::GAF::Phytochrome::PAS:: 70-186::219-404::415-595:: PAS::HisKA::HATPase_c 622-737::752-877::897-961::1011-1123 165 Mnom000110 PAS_2::GAF::Phytochrome::PAS:: 97-217::250-432::443-622:: PAS::HisKA::HATPase_c 653-769::784-906::926-990::1038-1150 166 Mnom000111 PAS_2::GAF::Phytochrome::PAS:: 97-217::250-432::443-622:: PAS::HisKA::HATPase_c 653-769::784-906::926-990::1038-1150 167 Mnom000112 PAS_2::GAF::Phytochrome::PAS:: 97-217::250-432::443-622:: PAS::HisKA::HATPase_c 653-769::784-906::926-990::1038-1150 168 Mnom000113, PAS_2::GAF::Phytochrome::PAS:: 70-186::219-404::415-595:: Mnom000116 PAS::HisKA::HATPase_c 622-737::752-877::897-961::1011-1123 169 Mnom000114, PAS_2::GAF::Phytochrome::PAS:: 97-217::250-432::443-622:: Mnom000117 PAS::HisKA::HATPase_c 653-769::784-906::926-990::1038-1150 170 Mnom000115, PAS_2::GAF::Phytochrome::PAS:: 97-217::250-432::443-622:: Mnom000118 PAS::HisKA::HATPase_c 653-769::784-906::926-990::1038-1150 171 Mnom000119 PAS_2::GAF::Phytochrome::PAS:: 70-186::219-404::415-595:: PAS::HisKA::HATPase_c 622-737::752-877::897-961::1011-1123 172 Mnom000738 DUF260  7-108 175 Mnom000857, DUF231 364-542 Mnom000858, Mnom000860 176 Mnom000873 Glyco_hydro_18  27-306 177 Mnom000886 Synaptobrevin 132-238 178 Mnom000923-Mnom000924 PAS_2::GAF::Phytochrome::PAS:: 97-217::250-432::443-622:: PAS::HisKA::HATPase_c 653-769::784-906::926-990::1038-1150 179 Mnom000937 PAS_3::PAS::Pkinase 141-227::390-504::582-870 180 Mnom000942, TP6A_N  89-156 Mnom000955-Mnom000956 181 Mnom000985 Response_reg  16-128 183 Mnom001055 Peptidase_S10  42-455 184 Mnom001090-Mnom001092 Gln-synt_N::Gln-synt_C 31-111::117-369 185 Mnom001138 UDPGP  26-443 186 Mnom001140 DnaJ 11-80 187 Mnom001141 DnaJ 12-78 188 Mnom001142 NAPRTase 175-439 189 Mnom001146 DUF296 114-234 190 Mnom001147 DUF296 114-234 191 Mnom001160 zf-A20::zf-AN1 14-38::115-155 192 Mnom001162 Cellulose_synt  284-1107 193 PHE0000597 Mannitol_dh::Mannitol_dh_C 10-132::157-383 194 PHE0001556 RimK::Mur_ligase_M::Mur_ligase_C 226-486::498-711::735-821 195 PHE0001557 RimK::Mur_ligase_M::Mur_ligase_C 217-370::491-704::728-813 196 PHE0001559 Peptidase_S51  52-212 198 PHE0003348 HLH 310-359 199 PHE0003706 SSF  54-471 200 PHE0003708 SSF  46-463 201 PHE0004230 E2F_TDP 227-292 202 PHE0004820 HLH 113-162 204 PHE0006017 Na_H_Exchanger  26-445 205 PHE0006102 Brix  27-342 207 PHE0006380 RNase_PH_C 129-201 209 PHE0006610 GSHPx 119-227 210 PHE0006613 GSHPx  29-137 211 PHE0006916 RAMP4  49-112 213 PHE0007643 LRRNT_2::LRR_1::LRR_1::LRR_1:: 38-79::155-177::179-201:: Pkinase_Tyr 227-249::429-696 216 PHE0008411 DUF778  41-162 218 PHE0008602 Gln-synt_N::Gln-synt_C 103-184::191-472 219 PHE0009339 HSP70  66-673 220 PHE0009341 TrkH 211-569 221 PHE0010195 PTR2  85-432 222 PHE0010352 CBFD_NFYB_HMF 31-96 223 PHE0010354 CBFD_NFYB_HMF 34-99 224 PHE0010646 Spermine_synth  46-291 225 PHE0010648 SNF2_N::Helicase_C 25-373::441-520 226 PHE0010649 SNF2_N::Helicase_C 24-374::442-521 227 PHE0010852 HLH 403-451 228 PHE0010956 Nucleoside_tran 167-450 230 PHE0011063 Globin  49-145 231 PHE0011543 zf-B_box  4-50 232 PHE0013202 HLH 172-221 233 PHE0013203 SAM_1::DRMBL 237-300::609-707 235 PHE0013292 PDZ::Peptidase_S41 220-304::340-503 238 PHE0013734 SRF-TF  9-59 239 TRG0000049 CBFD_NFYB_HMF  75-140 241 TRG0000332 CBFB_NFYA 192-248 242 1071693 PAS_2::GAF::Phytochrome::PAS 98-218::251-433::444-623::654-738

TABLE 11 Description of Pfam domains Accession Gathering Pfam domain name number cutoff Domain description 14-3-3 PF00244.12 25.0000; 14-3-3 protein 1-cysPrx_C PF10417.1 36.0000; C-terminal domain of 1-Cys peroxiredoxin 2OG-FeII_Oxy PF03171.12 11.5000; 2OG-Fe(II) oxygenase superfamily AhpC-TSA PF00578.13 4.8000; AhpC/TSA family Ald_Xan_dh_C PF01315.14 −7.6000; Aldehyde oxidase and xanthine dehydrogenase, a/b hammerhead domain Ald_Xan_dh_C2 PF02738.10 −233.0000; Aldehyde oxidase and xanthine dehydrogenase, molybdopterin binding domain AP2 PF00847.12 21.7000; AP2 domain AUX_IAA PF02309.8 −83.0000; AUX/IAA family Auxin_resp PF06507.5 25.0000; Auxin response factor B3 PF02362.13 26.5000; B3 DNA binding domain Brix PF04427.10 0.1000; Brix domain CBFB_NFYA PF02045.7 25.0000; CCAAT-binding transcription factor (CBF- B/NF-YA) subunit B CBFD_NFYB_HMF PF00808.15 18.4000; Histone-like transcription factor (CBF/NF-Y) and archaeal histone Cellulose_synt PF03552.6 −257.9000; Cellulose synthase CO_deh_flav_C PF03450.9 −1.0000; CO dehydrogenase flavoprotein C-terminal domain Copine PF07002.8 −36.5000; Copine DAO PF01266.16 −34.9000; FAD dependent oxidoreductase DnaJ PF00226.23 −8.0000; DnaJ domain DRMBL PF07522.6 25.0000; DNA repair metallo-beta-lactamase DUF231 PF03005.7 −46.3000; Arabidopsis proteins of unknown function DUF260 PF03195.6 −30.4000; Protein of unknown function DUF260 DUF296 PF03479.7 −3.0000; Domain of unknown function (DUF296) DUF761 PF05553.3 −97.7000; Cotton fibre expressed protein DUF778 PF05608.4 −38.6000; Protein of unknown function (DUF778) E2F_TDP PF02319.12 17.0000; E2F/DP family winged-helix DNA-binding domain Efhand PF00036.24 21.7000; EF hand FAD_binding_5 PF00941.13 −57.4000; FAD binding domain in molybdopterin dehydrogenase FAR1 PF03101.7 −12.7000; FAR1 DNA-binding domain FAT PF02259.15 −64.1000; FAT domain FATC PF02260.12 16.5000; FATC domain F-box PF00646.25 14.7000; F-box domain Fer2 PF00111.19 11.2000; 2Fe—2S iron-sulfur cluster binding domain Fer2_2 PF01799.12 −19.5000; [2Fe—2S] binding domain GAF PF01590.18 23.0000; GAF domain GATase_2 PF00310.13 −91.6000; Glutamine amidotransferases class-II Gln-synt_C PF00120.16 −124.0000; Glutamine synthetase, catalytic domain Gln-synt_N PF03951.11 9.0000; Glutamine synthetase, beta-Grasp domain Globin PF00042.14 −7.3000; Globin Glu_syn_central PF04898.6 25.0000; Glutamate synthase central domain Glu_synthase PF01645.9 −198.4000; Conserved region in glutamate synthase Glyco_hydro_18 PF00704.20 13.0000; Glycosyl hydrolases family 18 Glyco_transf_20 PF00982.13 −243.6000; Glycosyltransferase family 20 Glycos_transf_1 PF00534.12 −7.3000; Glycosyl transferases group 1 GMC_oxred_N PF00732.11 −71.1000; GMC oxidoreductase GSHPx PF00255.11 −16.0000; Glutathione peroxidase GXGXG PF01493.11 5.0000; GXGXG motif HATPase_c PF02518.18 22.4000; Histidine kinase-, DNA gyrase B-, and HSP90-like ATPase HEAT PF02985.14 16.4000; HEAT repeat Helicase_C PF00271.23 6.9000; Helicase conserved C-terminal domain HisKA PF00512.17 22.1000; His Kinase A (phosphoacceptor) domain HLH PF00010.18 8.3000; Helix-loop-helix DNA-binding domain HSP20 PF00011.13 13.0000; Hsp20/alpha crystallin family HSP70 PF00012.12 −286.0000; Hsp70 protein LRR_1 PF00560.25 10.9000; Leucine Rich Repeat LRRNT_2 PF08263.4 18.6000; Leucine rich repeat N-terminal domain Mannitol_dh PF01232.15 −10.5000; Mannitol dehydrogenase Rossmann domain Mannitol_dh_C PF08125.5 −79.1000; Mannitol dehydrogenase C-terminal domain Mur_ligase_C PF02875.13 8.0000; Mur ligase family, glutamate ligase domain Mur_ligase_M PF08245.4 −44.7000; Mur ligase middle domain Na_H_Exchanger PF00999.13 −67.9000; Sodium/hydrogen exchanger family NAPRTase PF04095.8 −88.5000; Nicotinate phosphoribosyltransferase (NAPRTase) family Nucleoside_tran PF01733.10 −85.0000; Nucleoside transporter PALP PF00291.17 −70.0000; Pyridoxal-phosphate dependent enzyme PAS PF00989.16 0.0000; PAS fold PAS_2 PF08446.3 −2.1000; PAS fold PAS_3 PF08447.3 17.2000; PAS fold PAS_4 PF08448.2 16.4000; PAS fold PDZ PF00595.16 20.7000: PDZ domain (Also known as DHR or GLGF) Peptidase_S10 PF00450.14 −198.0000; Serine carboxypeptidase Peptidase_S41 PF03572.10 −25.8000; Peptidase family S41 Peptidase_S51 PF03575.9 −11.1000; Peptidase family S51 PHD PF00628.21 26.2000; PHD-finger Phytochrome PF00360.12 13.0000; Phytochrome region PI3_PI4_kinase PF00454.19 14.8000; Phosphatidylinositol 3- and 4-kinase Pkinase PF00069.17 70.3000; Protein kinase domain Pkinase_Tyr PF07714.9 −155.0000; Protein tyrosine kinase PPR PF01535.12 1.6000; PPR repeat PTR2 PF00854.13 −50.0000; POT family RAMP4 PF06624.4 25.0000; Ribosome associated membrane protein RAMP4 Rapamycin_bind PF08771.3 25.0000; Rapamycin binding domain Redoxin PF08534.2 8.9000; Redoxin ResIII PF04851.7 0.8000; Type III restriction enzyme, res subunit Response_reg PF00072.16 4.1000; Response regulator receiver domain RimK PF08443.3 −56.1000; RimK-like ATP-grasp domain RNase_PH_C PF03725.7 20.0000; 3′ exoribonuclease family, domain 2 SAM_1 PF00536.22 11.3000; SAM domain (Sterile alpha motif) SAM_2 PF07647.9 0.0000; SAM domain (Sterile alpha motif) SAP PF02037.19 1.5000; SAP domain SNF2_N PF00176.15 −72.0000; SNF2 family N-terminal domain Spermine_synth PF01564.9 −93.8000; Spermine/spermidine synthase SRF-TF PF00319.10 11.0000; SRF-type transcription factor (DNA-binding and dimerisation domain) SSF PF00474.9 −162.3000; Sodium:solute symporter family Synaptobrevin PF00957.13 25.0000; Synaptobrevin TP6A_N PF04406.6 25.0000; Type IIB DNA topoisomerase Trehalose_PPase PF02358.8 −49.4000; Trehalose-phosphatase TrkH PF02386.8 −96.5000; Cation transport protein UDPGP PF01704.10 −265.2000; UTP--glucose-1-phosphate uridylyltransferase XRN_N PF03159.10 25.0000; XRN 5′-3′ exonuclease N-terminus zf-A20 PF01754.8 25.0000; A20-like zinc finger zf-AN1 PF01428.8 0.0000; AN1-like Zinc finger zf-B_box PF00643.16 15.3000; B-box zinc finger zf-CCHC PF00098.15 17.9000: Zinc knuckle zf-MIZ PF02891.12 −2.2000; MIZ/SP-RING zinc finger 

1. A recombinant DNA construct comprising a polynucleotide encoding a protein that has an amino acid sequence having at least 95% identity over at least 95% of the length of a reference sequence selected from the group consisting of SEQ ID NO: 122-242 when said amino acid sequence is aligned with said reference sequence.
 2. A recombinant DNA construct comprising a promoter that is functional in a plant cell and that is operably linked to a polynucleotide that, when expressed in a plant cell: (a) encodes a protein: i) having an amino acid sequence selected from the group consisting of SEQ ID NO: 123-140, 145-151, 153-173, 175-187, 189, 191-202, 204-207, 209-238, and 242; ii) having an amino acid sequence having at least 95% identity over at least 95% of a reference sequence selected from the group consisting of SEQ ID NO: 123-140, 145-151, 153-173, 175-187, 189, 191-202, 204-207, 209-238, and 242 when said amino acid sequence is aligned to said reference sequence; or iii) that is a homolog of a protein with an amino acid sequence selected from the group consisting of SEQ ID NO: 123-140, 145-151, 153-173, 175-187, 189, 191-202, 204-207, 209-238, and 242; or (b) is transcribed into an RNA molecule that suppresses the level of an endogenous protein in said plant cell wherein said endogenous protein has an amino acid sequence selected from the group consisting of SEQ ID NO: 122, 141-144, 152, 174, 188, 190, 203, 208, or 239-241 or is a homolog thereof; wherein said construct is stably integrated into plant chromosomal DNA.
 3. A transgenic plant cell comprising the recombinant DNA construct of claim 2 wherein said plant cell is in a plant selected by screening a population of transgenic plants that have been transformed with said construct for an enhanced trait as compared to control plants; and wherein said enhanced trait is enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein or enhanced seed oil.
 4. A mixture comprising plant cells of claim 3 and an antibody to a protein produced in said cells wherein said protein has an amino acid sequence that has at least 95% identity over at least 95% of the length of a reference sequence selected from the group consisting of SEQ ID NO: 122-242 when said amino acid sequence is aligned to said reference sequence.
 5. The plant cell of claim 3 further comprising DNA expressing a protein that provides tolerance from exposure to an herbicide comprising an agent applied at levels that are lethal to a wild type of said plant cell.
 6. The plant cell of claim 5 wherein the agent of said herbicide is a glyphosate, dicamba, or glufosinate compound.
 7. A transgenic plant comprising a plurality of plant cells of claim
 3. 8. The transgenic plant of claim 7 which is homozygous for said recombinant DNA.
 9. A transgenic seed comprising a plurality of plant cells of claim
 3. 10. The transgenic seed of claim 9 from a corn, soybean, cotton, canola, alfalfa, wheat, rice, sugarcane, or sugar beet plant.
 11. Grain comprising transgenic seed identifiable by the recombinant DNA construct of claim
 2. 12. Seed meal produced from transgenic seed identifiable by the recombinant DNA construct of claim
 2. 13. A transgenic pollen grain comprising a haploid derivative of a plant cell nucleus having a chromosome comprising the recombinant DNA construct of claim
 2. 14. A method for manufacturing non-natural, transgenic seed that can be used to produce a crop of transgenic plants with an enhanced trait resulting from expression of the stably-integrated, recombinant DNA construct of claim 2, said method comprising: (a) screening a population of plants for said enhanced trait and said recombinant DNA, wherein individual plants in said population exhibit said trait at a level less than, essentially the same as or greater than the level that said trait is exhibited in control plants which do not contain said recombinant DNA, wherein said enhanced trait is selected from the group of enhanced traits consisting of enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil; (b) selecting from said population one or more plants that exhibit said trait at a level greater than the level that said trait is exhibited in control plants, and (c) collecting seed from selected plant from step b.
 15. The method of claim 14 wherein said method for manufacturing said transgenic seed further comprises: (a) verifying that said recombinant DNA is stably integrated in said selected plants, and (b) analyzing tissue of said selected plant to determine the expression or suppression of a protein having the function of a protein having an amino acid sequence selected from the group consisting of one of SEQ ID NOs: 122-242.
 16. The method of claim 15 wherein said seed is corn, soybean, cotton, canola, alfalfa, wheat, rice, sugarcane, or sugar beet seed.
 17. A method of producing hybrid corn seed comprising: (a) acquiring hybrid corn seed from an herbicide tolerant corn plant which also has the stably-integrated, recombinant DNA construct of claim 2; (b) producing corn plants from said hybrid corn seed, wherein a fraction of the plants produced from said hybrid corn seed is homozygous for said recombinant DNA, a fraction of the plants produced from said hybrid corn seed is hemizygous for said recombinant DNA, and a fraction of the plants produced from said hybrid corn seed has none of said recombinant DNA; (c) selecting corn plants which are homozygous and hemizygous for said recombinant DNA by treating with an herbicide; (d) collecting seed from herbicide-treated-surviving corn plants and planting said seed to produce further progeny corn plants; (e) repeating steps (c) and (d) at least once to produce an inbred corn line; and (f) crossing said inbred corn line with a second corn line to produce hybrid seed. 